Biomedical subjects

B E Kream

Publications and source records attributed to B E Kream.

At least 37 records · Page 2Linked to original sources

PubMed1994

Regulation of the alpha 1(I) collagen promoter in vascular smooth muscle cells. Comparison with other alpha 1(I) collagen-producing cells in transgenic animals and cultured cells.

We have previously reported that the expression of the ColCAT3.6 transgene containing 3.5 kilobases (kb) of alpha 1(I) collagen (COL1A1) promoter sequence fused to the chloramphenicol acetyltransferase (CAT) reporter gene paralleled the expression of the endogenous gene in several connective tissues. We report here that the activity of the reporter gene in aorta from 7-day-old transgenic mice is 10-64-fold lower than in tendon or bone, whereas the endogenous gene is highly expressed in all three tissues. In contrast, the COL1A1 minigene containing 2.3 kb of upstream sequence, the first five exon/intron units, the last six exon/intron units, and 2 kb of 3'-flanking sequence showed high CAT activity in aorta. These results suggest that cis sequences found in ColCAT3.6 mediate high levels of COL1A1 expression in bone and tendon, but not in vascular smooth muscle cells (VSMC), whereas sequences located within the minigene, but not found in ColCAT3.6, mediate VSMC-specific expression. Analysis of promoter activity in cultured cells derived from transgenic tissues further suggests the presence of VSMC-specific regulatory domains. Transient transfection studies, however, failed to shows differential regulation. These differences stress the importance of not relying exclusively on transient transfection data when mapping tissue-specific regulatory domains.

Animals↗

PubMed1994

Upstream regulatory elements necessary for expression of the rat COL1A1 promoter in transgenic mice.

The activity of fusion genes containing fragments of the COL1A1 promoter was measured in tissues from 6- to 8-day-old transgenic mice. ColCAT3.6 contains approximately 3.6 kb (-3521 to 115 bp) of the rat COL1A1 gene, the chloramphenicol acetyltransferase (CAT) reporter gene, and the SV40 splice and polyadenylation sequences. ColCAT2.3 and ColCAT1.7 are deletion constructs that contain 2296 and 1667 bp of COL1A1 upstream from the RNA start site, respectively. For each transgene, up to six lines of mice were characterized. Both ColCAT3.6 and ColCAT2.3 had similar activity in bone and tooth; ColCAT1.7 was inactive. In transgenic calvariae, levels of transgene mRNA paralleled levels of CAT activity. In tendon, the activity of ColCAT2.3 was 3- to 4-fold lower than that of ColCAT3.6, and the activity ColCAT1.7 was 16-fold lower than that of ColCAT2.3. There was little activity of the ColCAT constructs in liver and brain. These data show that DNA sequences between -2.3 and -1.7 kb are required for COL1A1 promoter expression in bone and tooth; sequences that control expression in tendon are distributed between -3.5 and -1.7 kb of the promoter, with sequences downstream of -1.7 kb still capable of directing expression to this tissue. The cis elements that govern basal expression of COL1A1 in transgenic calvariae appear to be different from those required for optimal expression of the COL1A1 promoter in stably transfected osteoblastic cells.

Animals↗

PubMed1994

Analysis of regulatory regions in the COL1A1 gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression in osteoblastic cells.

The synthesis of type I collagen in bone cells is inhibited by the calcium-regulating hormone 1,25-dihydroxyvitamin D3. Earlier work from our laboratories has indicated that vitamin D regulation is at the level of transcription, based on results from both nuclear run-off assays and functional promoter analysis of a hybrid gene consisting of a 3.6 kb COL1A1 promoter fragment fused to the chloramphenicol acetyltransferase reporter gene. In the present study, we investigated the molecular basis for vitamin D-mediated transcriptional repression of the COL1A1 gene and report the identification of a region within the COL1A1 upstream promoter (the HindIII-Pstl restriction fragment between nucleotides -2295 and -1670) which is necessary for 1,25-dihydroxyvitamin D3 responsiveness in osteoblastic cells. This hormone-mediated inhibitory effect on the marker gene parallels the inhibition of the endogenous collagen gene. A 41 bp fragment from this region (between nucleotides -2256 and -2216) contains a sequence which is very similar to vitamin D-responsive elements identified in the osteocalcin gene. Extracts from cultured cells which express a high level of vitamin D receptor contain a hormone:receptor complex that binds specifically to this 41 bp fragment, as demonstrated by bandshift analysis. However, deletion of this vitamin D receptor binding region from either a -3.5 kb or a -2.3 kb promoter fragment did not abolish vitamin D responsiveness. These results indicate that a vitamin D response element similar to that described for other vitamin D responsive genes (osteocalcin and osteopontin) does not alone mediate the repression of COL1A1 by 1,25-dihydroxyvitamin D3.

3T3 Cells↗

PubMed1993

Basic fibroblast growth factor inhibits type I collagen gene expression in osteoblastic MC3T3-E1 cells.

We examined the effect of basic fibroblast growth factor (bFGF) on alpha 1(I) procollagen mRNA levels, alpha 1(I) collagen gene transcription, and alpha 1(I) collagen promoter activity in osteoblastic MC3T3-E1 cells. Cells were stably transfected with ColCAT 3.6, containing 3521 base pairs of alpha 1(I) collagen promoter DNA, fused to the CAT reporter gene, or an upstream deletion mutant of ColCAT 3.6 designated ColCAT 2.3. After 48 h, bFGF (0.1-10 nM) inhibited the incorporation of [3H]proline into collagenase-digestible protein (CDP). Indomethacin did not alter the inhibitory effect of bFGF on CDP labeling. Aphidicolin, an inhibitor of DNA synthesis, did not block the inhibitory effect of bFGF on CDP. bFGF (1-10 nM) decreased alpha 1(I) procollagen mRNA levels, with maximal inhibition, nearly 99% of control, caused by 10 nM bFGF. After 48 h, bFGF (1 nM) reduced alpha 1(I) procollagen gene transcription by about 92%. ColCAT 3.6 activity was inhibited with 0.1-10 nM bFGF and was maximally repressed by about 83% with 10 nM bFGF. In contrast, bFGF (1 and 10 nM) caused a stimulation of ColCAT 2.3 activity. These data show that bFGF inhibits collagen synthesis by a transcriptional mechanism and the alpha 1(I) collagen promoter contains DNA sequences which mediate bFGF inhibition of type I collagen gene expression in bone.

3T3 Cells↗

PubMed1993

Transgenic expression of COL1A1-chloramphenicol acetyltransferase fusion genes in bone: differential utilization of promoter elements in vivo and in cultured cells.

To directly compare the patterns of collagen promoter expression in cells and tissues, the activity of COL1A1 fusion genes in calvariae of neonatal transgenic mice and in primary bone cell cultures derived by sequential digestion of transgenic calvariae was measured. ColCAT3.6 contains 3.6 kb (positions -3521 to +115) of the rat COL1A1 gene ligated to the chloramphenicol acetyltransferase (CAT) reporter gene. ColCAT2.3 and ColCAT1.7 are 5' deletion mutants which contain 2,296 and 1,672 bp, respectively, of COL1A1 DNA upstream from the transcription start site. ColCAT3.6 activity was 4- to 6-fold lower in primary bone cell cultures than in intact calvariae, while ColCAT2.3 activity was at least 100-fold lower in primary bone cells than in calvariae. These changes were accompanied by a threefold decrease in collagen synthesis and COL1A1 mRNA levels in primary bone cells compared with collagen synthesis and COL1A1 mRNA levels in freshly isolated calvariae. ColCAT3.6 and ColCAT2.3 activity was maintained in calvariae cultured in the presence or absence of serum for 4 to 7 days. Thus, when bone cells are removed from their normal microenvironment, there is parallel downregulation of collagen synthesis, collagen mRNA levels, and ColCAT3.6 activity, with a much greater decrease in ColCAT2.3. These data suggest that a 624-bp region of the COL1A1 promoter between positions -2296 and -1672 is active in intact and cultured bone but inactive in cultured cells derived from the bone. We suggest that the downregulation of COL1A1 activity in primary bone cells may be due to the loss of cell shape or to alterations in cell-cell and/or cell-matrix interactions that normally occur in intact bone.

Animals↗

PubMed1993

Effects of prostaglandin E2 on bone formation in cultured fetal rat calvariae: role of insulin-like growth factor-I.

Prostaglandin E2 (PGE2) can stimulate collagen synthesis in bone at low concentrations or in the presence of cortisol. Moreover, cortisol inhibits and PGE2 stimulates the production of insulin-like growth factor (IGF-I) in cultured osteoblastic cells. Therefore, we examined the role of IGF-I in the response to PGE2. In 96-h fetal rat calvarial organ cultures, PGE2 increased, and cortisol and indomethacin decreased the medium IGF-I concentration, suggesting that both exogenous and endogenous PGs regulate IGF-I production. In the presence of cortisol, the stimulatory effects of PGE2 on medium IGF-I and incorporation of [3H] proline into collagenase-digestible protein were highly correlated (r = 0.95). When exogenous IGF-I (30 nM) was added, the stimulatory effect of PGE2 was abrogated in the absence, but not the presence, of cortisol. When we added IGF-binding proteins, which blocked the effects of IGF-I and IGF-II, collagenase-digestible protein labeling was decreased in control and cortisol-treated cultures, whereas the stimulatory effect of PGE2 was reduced, but not abrogated. We conclude that endogenous IGFs play a role in maintaining bone formation in cultured fetal rat calvariae and may mediate in part the anabolic response to PGE2. However, the PGE2 response probably involves additional IGF-independent pathways.

Animals↗

PubMed1993

Prostaglandin E2 inhibits alpha 1(I)procollagen gene transcription and promoter activity in the immortalized rat osteoblastic clonal cell line Py1a.

High concentrations of prostaglandin E2 (PGE2) inhibit collagen synthesis and reduce alpha 1(I)procollagen messenger RNA (mRNA) levels in cultured fetal rat calvariae. To examine the mechanism of this effect, we used the immortalized rat osteoblastic clonal cell line, Py1a. PGE2 at 1 microM inhibited the incorporation of [3H]proline into collagenase-digestible protein (CDP) and increased incorporation into noncollagen protein, whereas 0.1 microM PGE2 increased both CDP and noncollagen protein labeling. Because insulin-like growth factor-I (IGF-I) is an anabolic hormone in bone and PGE2 can increase its production, we added exogenous IGF-I (10 nM) to Py1a cultures. In the presence of IGF-I, PGE2 from 10 nM to 1 microM had only an inhibitory effect on CDP labeling and alpha 1(I)procollagen mRNA levels. PGE2 at 1 microM decreased the rate of alpha 1(I)procollagen gene transcription in the presence or absence of IGF-I, determined by a nuclear run-on assay. Py1a cells were stably transfected with chimeric genes containing varying lengths of the 5'-upstream region of the rat alpha 1(I)procollagen promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene. In cells transfected with ColCAT 3.6, which contains 3520 base pairs of 5'-upstream DNA, CAT activity was inhibited by PGE2, but the inhibition was less than that observed for CDP labeling. With smaller 5'-upstream regions, there was no inhibitory effect of PGE2. These results demonstrate that PGE2 inhibits alpha 1(I)procollagen gene transcription and the activity of a region between -3.5 and -2.3 kilobases of the 5'-upstream collagen gene promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Parathyroid hormone represses alpha 1(I) collagen promoter activity in cultured calvariae from neonatal transgenic mice.

We examined the effect of PTH on the activity of alpha 1(I) collagen promoter fusion genes in cultured calvariae from transgenic mice. The parent construct, ColCAT 3.6, contains 3520 basepairs of 5' rat alpha 1(I) collagen DNA, 115 basepairs of untranslated alpha 1(I) collagen-coding DNA, and the bacterial chloramphenicol acetyltransferase reporter gene, while the 5'-deletion ColCAT 2.3 contains 2296 kilobases of rat alpha 1(I) collagen promoter sequence. Transgenic mouse lines harboring these collagen promoter fusion genes were developed using the oocyte microinjection technique, and for each construct, three different lines of mice were tested. Calvariae from 6- to 8-day-old transgenic mice were cultured for 48 h with or without bovine PTH-(1-34). ColCAT 3.6 and ColCAT 2.3 were expressed at comparable levels in calvariae and were inhibited by PTH. There were parallel decreases in the incorporation of [3H]proline into collagen and levels of the endogenous alpha 1(I) collagen mRNA and transgene mRNA. Forskolin at 10 microM mimicked the inhibitory effect of PTH on promoter activity in ColCAT 3.6 and ColCAT 2.3 calvariae. A RNase protection assay showed that the transgene was initiated correctly from the transgene promoter. These data show that PTH and cAMP can repress collagen promoter activity in calvariae from transgenic mice, suggesting that the alpha 1(I) collagen promoter may contain cis elements down-stream of -2.3 kilobases that mediate PTH and cAMP repression of collagen gene expression in bone. Cultured bone explants from transgenic mice can be used as a model to study hormonal regulation of alpha 1(I) collagen promoter constructs.

Animals↗

PubMed1992

Differential utilization of regulatory domains within the alpha 1(I) collagen promoter in osseous and fibroblastic cells.

Type I collagen is expressed in a variety of connective tissue cells and its transcriptional regulation is highly complex because of the influence of numerous developmental, environmental, and hormonal factors. To investigate the molecular basis for one aspect of this complex regulation, the expression of alpha 1(I) collagen (COL1A1) gene in osseous tissues, we fused a 3.6-kb DNA fragment between bases -3,521 and +115 of the rat COL1A1 promoter, and three deletion mutants, to the chloramphenicol acetyltransferase (CAT) marker gene. The expression of these ColCAT transgenes was measured in stably transfected osteoblastic cell lines ROS 17/2.8, Py-la, and MC3T3-E1 and three fibroblastic lines NIH-3T3, Rat-1, and EL2. Deletion of the distal 1.2-kb fragment of the full-length ColCAT 3.6 construct reduced the promoter activity 7- to 30-fold in the osteoblastic cell lines, twofold in EL2 and had no effect in NIH-3T3 and Rat-1 cells. To begin to assess the function of COL1A1 upstream regulatory elements in intact animals, we established transgenic mouse lines and examined the activity of the ColCAT3.6 construct in various tissues of newborn animals. The expression of this construct followed the expected distribution between the high and low collagen-producing tissues: high levels of CAT activity in calvarial bone, tooth, and tendon, a low level in skin, and no detectable activity in liver and brain. Furthermore, CAT activity in calvarial bone was three- to fourfold higher than that in the adjacent periosteal layer. Immunostaining for CAT protein in calvaria and developing tooth germ of ColCAT3.6 mice also confirmed the preferred expression of the transgene in differentiated osteoblasts and odontoblasts compared to fibroblast-like cells of periosteum and dental papilla. This study suggests that the 3.6-kb DNA fragment confers the strong expression of COL1A1 gene in high collagen producing tissues of intact animals and that the 5' flanking promoter sequence between -3,521 and -2,295 bp contains one or more stimulatory elements which are preferentially active in osteoblastic cells.

3T3 Cells↗

PubMed1991

The role of DNA synthesis in the responses of fetal rat calvariae to cortisol.

To determine the extent to which the effects of cortisol on collagen synthesis in 21 day fetal rat calvariae are linked to its effects on cell replication, calvariae were cultured for 24-72 h with 0.1 and 1 microM cortisol in the presence or absence of 1 mM hydroxyurea (HU) or 30 microM aphidicolin (APC), inhibitors of DNA synthesis. The incorporation of [3H]proline into collagenase-digestible protein (CDP) and [3H]thymidine into DNA were measured during the last 2 h of culture. At 24 h HU and APC decreased thymidine incorporation by greater than 90%, and this remained low for the duration of culture. In contrast, cortisol reduced thymidine incorporation by only 44% at 72 h. Although cortisol caused a 24 h stimulatory effect and a 48 and 72 h inhibitory effect on CDP labeling and the percentage of collagen being synthesized (PCS), HU, and APC had no effect on basal CDP labeling or PCS over the 72 h culture period. Cortisol caused parallel alterations in the steady-state levels of alpha-1(I) procollagen mRNA, suggesting that its effects occur at the pretranslational level. At 24 h HU and APC did not prevent the stimulatory effect of cortisol on CDP labeling and PCS. At 48 h the inhibitory effects of cortisol on CDP labeling and PCS were observed in the presence of APC but not in the presence of HU. At 72 h the inhibitory effects of cortisol on CDP labeling and PCS were still observed in the presence of HU and APC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Structural determinants of the capacity of heparin to inhibit collagen synthesis in 21-day fetal rat calvariae.

Earlier work from our laboratory demonstrated that heparin inhibits type I collagen and DNA synthesis in fetal rat calvariae in vitro. In this paper we have analyzed the structural features of heparin that determine its inhibitory effect on collagen synthesis. These experiments were performed using unmodified heparins and low-molecular-weight heparins from different manufacturers, nonheparin glycosaminoglycans, desulfated heparins, anticoagulant and nonanticoagulant heparin, and chemically defined heparin oligosaccharides. Low-molecular-weight heparin (Mr 3700-5100) inhibited collagen synthesis, but oligosaccharides (disaccharides to decasaccharide, Mr 665-3000) did not. The glycosaminoglycans chondroitin sulfate B, heparan sulfate, and hyaluronic acid did not alter collagen synthesis but dextran sulfate was as inhibitory as unmodified heparin. Nonanticoagulant as well as anticoagulant low-molecular-weight heparin fractions inhibited collagen synthesis. Modification of heparin by total desulfation, O-desulfation, or N-desulfation and re-N-acetylation resulted in the loss of inhibitory property, suggesting that the degree of sulfation contributed to heparin's inhibitory effect. Low-molecular-weight heparins from different manufacturers were just as inhibitory as native heparin on collagen synthesis. We therefore conclude that low-molecular-weight heparin compounds offer no protection against heparin-induced osteoporosis. Our findings also suggest that the size and sulfation of a heparin-derived oligosaccharide contribute to its ability to inhibit collagen synthesis in bone.

Animals↗

PubMed1990

Effect of heparin on bone formation in cultured fetal rat calvaria.

To assess the effects of heparin on bone formation we measured [3H]proline incorporation into collagenase-digestible (CDP) and noncollagen protein (NCP), [3H]thymidine (TdR) incorporation into DNA, and DNA content in 21-day-old fetal rat calvaria cultured in BGJ medium with bovine serum albumin for 24-96 hours. Heparin at 5-125 micrograms/ml decreased TdR incorporation by 26-51% at 24 and 96 hours. At 96 hours, heparin 5, 25, and 125 micrograms/ml decreased [3H]proline incorporation into CDP by 41, 48, and 32%, respectively, with no significant change in NCP. To evaluate the possible role of PGE2 in these inhibitory responses, media PGE2 concentration was measured and the effects of heparin on CDP labeling and DNA synthesis were tested in the presence of indomethacin, piroxicam, and flurbiprofen to inhibit endogenous prostaglandin E2 (PGE2) production and in the presence of a high concentration (10(-7) M) of exogenous PGE2. Heparin did not alter PGE2 production at 24 hours but at 48 hours there was a significant reduction. At 96 hours, indomethacin (10(-6) M) inhibited [3H]-proline incorporation into CDP by 38% but had no effect on the labeling of NCP. Heparin had no further significant inhibitory effect in the presence of indomethacin. Piroxicam and flurbiprofen did not alter DNA content and had a smaller inhibitory effect than indomethacin on the labeling of CDP. Moreover, addition of heparin produced a further inhibition of CDP and DNA content and finally, heparin decreased CDP labeling by 71% in the presence of PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Effects of ethanol and acetaldehyde on collagen synthesis, prostaglandin release and resorption of fetal rat bone in organ culture.

We tested the effect of ethanol and its metabolite, acetaldehyde, on bone formation as measured by [3H]proline incorporation into collagenase digestible protein (CDP) and noncollagen protein (NCP), and on DNA synthesis as measured by [3H]thymidine (TdR) incorporation in fetal rat calvaria. We also determined the effects of ethanol and acetaldehyde on prostaglandin E2 (PGE2) release from calvaria and on bone resorption as measured by 45Ca release from fetal rat long bones. Bones were cultured in multiwell plastic dishes (open system) or in stoppered Erlenmeyer flasks (closed system) for 24 to 96 h. In the open system, 1% ethanol (v/v; 172 mM) resulted in a 31% decrease in TdR incorporation at 24 h with no effect on CDP and NCP. At 0.1% (17.2 mM), ethanol increased TdR by 22%, CDP by 73% and NCP by 67% at 24 h, but these effects were not sustained at 96 h. At 24 h, 1% and 0.3% ethanol decreased PGE2 release by 88% and 75% respectively. This effect was sustained for 96 h only at the higher concentration. In the closed system, 0.1% ethanol increased TdR incorporation by 38% at 24 h. However, there was no effect on the labeling of CDP or NCP. Because its boiling point is 21 degrees C, acetaldehyde could only be tested in the closed system. Acetaldehyde markedly inhibited bone metabolism. At 24 h, 0.003% (0.54 mM) to 0.01% (1.79 mM) acetaldehyde caused a dose-related inhibition of TdR incorporation from 23 to 45%. At 0.01% and 0.03% acetaldehyde inhibited proline incorporation into CDP by 48% and 94% and NCP by 40% and 74% respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde↗

PubMed1990

Parathyroid hormone blocks the stimulatory effect of insulin-like growth factor-I on collagen synthesis in cultured 21-day fetal rat calvariae.

We examined the interaction of parathyroid hormone (PTH) and recombinant human insulin-like growth factor I (IGF-I) on collagen synthesis in 21-day fetal rat calvariae as assessed by measuring the incorporation of [3H]proline into collagenase-digestible protein. After 96 hours of culture, 10 nM PTH antagonized the stimulation of collagen synthesis and partially blocked the increase in dry weight produced by 10 nM IGF-I. The effect of PTH to block IGF-I stimulated collagen synthesis was observed in the central bone of calvariae and was mimicked by forskolin and phorbol 12-myristate 13-acetate, but not by 1,25-dihydroxyvitamin D3, transforming growth factor-alpha or dexamethasone. Our data are consistent with the concept that the direct effect of PTH is to inhibit basal CDP labeling and fully oppose IGF-I stimulated CDP labeling. The finding that this effect of PTH is mimicked by forskolin and PMA suggests that this block in IGF-I stimulation of CDP labeling involves both cAMP and protein kinase C mediated pathways.

Animals↗

PubMed1990

Cortisol enhances the anabolic effects of insulin-like growth factor I on collagen synthesis and procollagen messenger ribonucleic acid levels in cultured 21-day fetal rat calvariae.

We examined the ability of cortisol to modulate the stimulatory effects of recombinant human insulin-like growth factor-I (IGF-I) on collagen synthesis, procollagen messenger RNA (mRNA) levels and DNA synthesis in 21-day fetal rat calvariae maintained in serum-free organ culture for 24-96 h. Collagen synthesis was quantitated by measuring the incorporation of [3H]proline into collagenase-digestible protein (CDP) and alpha-1(I) procollagen mRNA transcripts were assessed by Northern blot analysis. Cell replication was quantitated by measuring the incorporation of [3H]thymidine into bone. As described previously, 100 nM cortisol had a biphasic effect on CDP labeling, increasing CDP after 24 h and decreasing CDP after 48, 72, and 96 h of culture. IGF-I alone increased CDP labeling by 1.6-fold after 24 h and by 2-fold after 48 or 72 h of culture, and cortisol potentiated this anabolic effect. In the presence of 100 nM cortisol, IGF-I increased CDP labeling by 2.6-fold after 24 h, by 5-fold after 48 h, and by 8-fold after 72 h of culture. A higher concentration of cortisol (1000 nM) also potentiated the IGF-I response on CDP labeling after 96 h of culture. In the presence of 100 nM cortisol, concentrations of IGF-I lower than 10 nM consistently increased CDP labeling and the percent collagen synthesized whereas these concentrations were not always effective without cortisol. PTH, which like cortisol decreased basal CDP labeling, did not enhance the stimulatory effects of IGF-I. Cortisol also enhance the stimulatory effects of IGF-I on alpha-1(I) procollagen mRNA levels indicating that the potentiation of CDP labeling occurs via a pretranslational mechanism. IGF-I had little effect on the incorporation of [3H]thymidine into bone except in the presence of cortisol. Nevertheless, the ability of cortisol to potentiate the stimulatory effect of IGF-I on CDP labeling was independent of cell replication since the enhancement persisted in the presence of aphidicolin, a DNA synthesis inhibitor. Our findings show that physiological concentrations of cortisol can modulate the responsiveness of cells within cultured fetal rat calvariae to the anabolic effects of exogenous IGF-I.

Animals↗

PubMed1990

Interleukin-1 alpha and phorbol ester inhibit collagen synthesis in osteoblastic MC3T3-E1 cells by a transcriptional mechanism.

The effects of recombinant human interleukin-1 alpha (IL-1) on procollagen gene expression were examined in the clonal mouse osteoblastic cell line MC3T3-E1. Cells were grown in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and 50 micrograms/ml ascorbic acid. Collagen synthesis was assessed as [3H]proline incorporation into collagenase-digestible protein (CDP). Procollagen mRNA levels were determined by Northern blot analysis using a 32P-labeled alpha 1(I) cDNA. Transcription rates were determined by nuclear run-off assay. IL-1 at 1-1000 pg/ml caused a concentration-dependent inhibition of CDP, which was maximally reduced by 75-80%, and a parallel reduction of procollagen alpha 1(I) mRNA levels. The effects of IL-1 were mimicked by the tumor promoter phorbol 12-myristate 13-acetate (PMA) at 1-100 nM, which inhibited CDP and reduced procollagen alpha 1(I) mRNA levels to a similar extent. The effects of IL-1 and PMA were independent of prostaglandin production, since indomethacin did not alter the inhibitory effect of either agent on CDP. Neither IL-1 (up to 10 ng/ml) nor PMA (100 nM) affected adenylate cyclase activity, while forskolin (10 microM), PTH (10 nM) and prostaglandin E2 (1 microM) stimulated adenylate cyclase activity 3- to 5-fold. However, forskolin (10 microM) and (Bu)2cAMP (100 microM) failed to alter CDP or procollagen alpha 1(I) mRNA levels. IL-1 (1 ng/ml) and PMA (100 nM) reduced transcription of the alpha 1(I) procollagen gene by 70% and 80%, respectively, while alpha 2(I) transcription was decreased by 59% and 53%. Neither IL-1 nor PMA affected transcription of the beta-actin or beta-tubulin genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Effects of transforming growth factor alpha and interleukin-1 on DNA synthesis, collagen synthesis, procollagen mRNA levels, and prostaglandin E2 production in cultured fetal rat calvaria.

Transforming growth factor alpha (TGF-alpha) and interleukin-1 (IL-1) have been shown to affect bone metabolism in vitro by prostaglandin-dependent and PG-independent mechanisms. We assessed the effects of the combination of these two agents on [3H]thymidine (TdR) incorporation into DNA, DNA content, [3H]proline incorporation into collagenase-digestible (CDP), noncollagen protein (NCP), and PGE2 production in 21 day fetal rat calvaria cultured for 24-96 h. We also determined whether TGF-alpha plus IL-1 altered procollagen mRNA levels at 96 h. TGF-alpha, 1-30 ng/ml, produced a 41-59% increase in TdR incorporation into DNA, but the effect was partially blocked by human recombinant IL-1. At 96 h TGF-alpha alone or in combination with IL-1 significantly increased the DNA content of calvaria. At 96 h, TGF-alpha inhibited CDP labeling and the addition of IL-1 further enhanced this inhibitory effect. The enhanced inhibitory effect of TGF-alpha plus IL-1 on collagen synthesis was associated with a synergistic increase in prostaglandin accumulation in the medium. Addition of indomethacin blocked PGE2 accumulation and partially reversed the inhibitory effect of TGF-alpha alone or in combination with IL-1 on collagen synthesis. TGF-alpha decreased procollagen mRNA levels by 55%, but the combination of TGF-alpha plus IL-1 decreased procollagen mRNA levels by 82%. Our results show that TGF-alpha and IL-1, which are both produced by certain tumors as well as activated macrophages, appear to act synergistically to increase prostaglandin synthesis and inhibit collagen synthesis in vitro. Thus these agents may have a regulatory role on bone formation in vivo.

Animals↗

PubMed1989

1,25-Dihydroxyvitamin D3 inhibits transcription of type I collagen genes in the rat osteosarcoma cell line ROS 17/2.8.

Our previous studies have demonstrated that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3)] reduces type I collagen synthesis and steady state levels of procollagen mRNA in cultured fetal rat calvaria and rat osteosarcoma cells. To determine whether 1,25-(OH)2D3 regulates transcription of type I collagen genes, transcription rates were measured directly in nuclei isolated from ROS 17/2.8 cells using a nuclear run-off assay. Transcription was allowed to proceed in the presence of [32P]UTP for 20 min, at which time incorporation of radiolabeled UTP into trichloroacetic acid-precipitable material was maximal. UTP incorporation was inhibited 90% by 3 micrograms/ml actinomycin-D and 40% by 1 microgram/ml alpha-amanitin. Treatment of ROS 17/2.8 cells with 1,25-(OH)2D3 inhibited procollagen gene transcription in a concentration and time dependent manner. Procollagen transcription was reduced by approximately 50% of the control rate by 10 nM 1,25-(OH)2D3, and this inhibition was maximal after 24 h of 1,25-(OH)2D3 treatment. The inhibition of procollagen transcription was specific for collagen, since total RNA synthesis and beta-actin transcription were not inhibited by 1,25-(OH)2D3. The magnitude of the decrease of procollagen transcription by 1,25-(OH)2D3 was comparable to its inhibition of steady state procollagen mRNA levels, suggesting that transcription is the predominant mechanism by which 1,25-(OH)2D3 regulates collagen gene expression in bone cells.

Animals↗