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B E Kream

Publications and source records attributed to B E Kream.

At least 55 records · Page 3Linked to original sources
PubMed1989

Insulin increases the steady state level of alpha-1(I) procollagen mRNA in the osteoblast-rich segment of fetal rat calvaria.

Insulin at 3-100 nM increased the steady state level of alpha-1(I) procollagen mRNA and stimulated collagen synthesis in the osteoblast-rich segment of central bone from 21-day-old fetal rat calvaria. The increases in the level of procollagen mRNA and the rate of collagen synthesis were observed 18 h after the addition of insulin to the cultures. The removal of insulin from calvaria incubated for 24 h with 3 nM insulin caused collagen synthesis and the level of alpha-1(I) procollagen mRNA to return to control values within 5 h. Adding insulin back to calvaria withdrawn from insulin treatment for 3 h did not rescue the decay in collagen synthesis or the level of alpha-1(I) procollagen mRNA. Insulin increased the steady state levels of alpha-1(I) procollagen mRNA in the presence of the RNA synthesis inhibitor actinomycin-D. Our data suggest that in fetal rat bone, one mechanism by which insulin increases the steady state level of alpha-1(I) procollagen mRNA may be by altering its stability.

Animals↗
PubMed1988

Inhibition of bone collagen synthesis by the tumor promoter phorbol 12-myristate 13-acetate.

We characterized the effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on osteoblast function and DNA synthesis in 21-day-old fetal rat calvaria maintained in organ culture. Protein synthesis was determined by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein (NCP), respectively. Alkaline phosphatase activity was assessed as the release of p-nitrophenol from p-nitrophenol phosphate. DNA synthesis was determined by the incorporation of [3H]thymidine into acid-insoluble bone and total DNA content. PMA at 3-100 ng/ml (4-133 nM) caused a dose-related inhibition of collagen synthesis that was observed 6 hours after adding PMA to calvaria. PMA inhibited collagen synthesis in the osteoblast-rich central bone of calvaria but did not alter collagen synthesis in the periosteum. There was little effect of PMA on noncollagen protein synthesis in the central bone or periosteum. Phorbol esters that do not promote tumor formation in vivo did not alter collagen synthesis in calvaria. PMA stimulated prostaglandin E2 (PGE2) production in calvaria, but indomethacin did not alter the inhibitory effect of PMA on bone collagen synthesis. PMA decreased alkaline phosphatase activity measured after 48 hr of culture and increased the incorporation of [3H]thymidine into bone and DNA content after 96 hr of culture. These data indicate that PMA inhibits collagen synthesis and alkaline phosphatase activity, while stimulating DNA synthesis, suggesting that activation of protein kinase C might regulate osteoblast function and bone cell replication.

Alkaline Phosphatase↗
PubMed1988

In situ hybridization analysis of the expression of the type II collagen gene in the developing chicken limb bud.

In situ hybridization with [32P]- or [35S]-labeled double-stranded DNA or single-stranded RNA probes was used to investigate the temporal and spatial distribution of cartilage-characteristic type II collagen mRNA during embryonic chick limb development and cartilage differentiation in vivo. When the type II collagen probes were hybridized to sections through embryonic limb buds at the earliest stages of their development (stages 18-25), an accumulation of silver grains representing type II collagen mRNA first became detectable in the proximal central core of the limb coincident with the prechondrogenic condensation of mesenchymal cells that characterizes the onset of cartilage differentiation. At later stages of development (stage 32; 7 days) intense hybridization signals with the type II collagen probes were localized over the well differentiated cartilage rudiments, whereas few or no silver grains above background were observed over the non-chondrogenic tissues. In contrast, sections hybridized with a probe complementary to mRNA for the alpha 1 chain of type I collagen exhibited an intense hybridization signal over the perichondrium and little or no signal over the cartilage primordia. At all stages of development examined, [32P]-labeled double-stranded DNA probes or single-stranded RNA probes labeled with either [32P] or [35S] provided adequate hybridization signals. Several experimental protocols were employed to control for the potential cross-hybridization and non-specific hybridization of the type II collagen probes. These included the utilization of labeled noncomplementary "sense-strand" type II collagen RNA as a control probe for nonspecific background, and prehybridization with a large excess of appropriate unlabeled RNA to block sequences in heterologous collagen RNAs that might cross-hybridize to the specific labeled probe.

Animals↗
PubMed1988

Variable effects of dexamethasone on protein synthesis in clonal rat osteosarcoma cells.

We examined the effects of dexamethasone on protein synthesis in clonal rat osteoblastic osteosarcoma (ROS) cell lines by measuring the incorporation of [3H]proline into collagenase-digestible and noncollagen protein in the cell layer and medium of the cultures. In ROS 17/2 and subclone C12 of ROS 17/2.8, dexamethasone decreased collagen synthesis with no change in DNA content of the cultures. In ROS 17/2.8 and its subclone G2, dexamethasone stimulated collagen and noncollagen protein synthesis, with a concomitant decrease in the DNA content of the cells. These data indicate that ROS cell lines are phenotypically heterogeneous and suggest that in normal bone there may be distinct subpopulations of osteoblasts with varying phenotypic traits with respect to the regulation of protein synthesis.

Animals↗
PubMed1986

Hormonal regulation of collagen synthesis in a clonal rat osteosarcoma cell line.

Collagen synthesis in rat osteosarcoma cell line 17/2 (ROS 17/2) was assessed by measuring the incorporation of [3H]proline into collagenase-digestible protein and the formation of [3H]hydroxyproline. PTH and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited collagen synthesis in ROS 17/2 cells in a time- and dose-dependent manner. PTH reduced collagen synthesis after a 3-h incubation, whereas the effect of 1,25-(OH)2D3 was somewhat slower. Maximal and half-maximal inhibition of collagen synthesis occurred at approximately 1 and 0.1 nM of each hormone, respectively. At confluency, ROS 17/2 cells synthesized 96% type I and 4% type III collagen. PTH reduced the synthesis of type I, but not type III, collagen. PTH and 1,25-(OH)2D3 also reduced procollagen mRNA levels, as determined by a dot blot hybridization assay. Thus, ROS 17/2 cells are a convenient model system for studying the hormonal regulation of collagen metabolism and gene expression in a cloned cell line with the osteoblastic phenotype.

Animals↗
PubMed1985

Characterization of the effect of insulin on collagen synthesis in fetal rat bone.

We characterized the effect of insulin on collagen synthesis in 21-day-old fetal rat calvaria maintained in organ culture. All experiments were done in the presence of 100 mg/dl glucose and 3 mM phosphate, which were found to be optimal concentrations for insulin responsiveness. All concentrations of insulin tested (1 nM to 1 microM) increased the percentage of collagen being synthesized in the central bone, whereas only high concentrations of hormone (100 nM to 1 microM) increased the percentage of collagen being synthesized in periosteum. Insulin at 3 nM increased the labeling of type I collagen in the central bone, but did not alter the labeling of type I or III collagen in the periosteum. Proinsulin was approximately 10-100 times less effective than insulin in stimulating collagen synthesis, whereas porcine relaxin and C-peptide were ineffective. Insulin did not enhance the deposition of newly synthesized collagen in the bone by a mechanism that involved decreasing the degradation of collagen. To determine whether insulin enhanced collagen synthesis by increasing the replication of collagen-synthesizing cells, we tested the effect of insulin in the presence of hydroxyurea, a DNA synthesis inhibitor. Hydroxyurea at 1 mM had little effect on collagen synthesis in control cultures or those treated with 1 or 10 nM insulin. However, hydroxyurea blunted the stimulation of collagen synthesis that occurred at higher concentrations of insulin. These experiments suggest that insulin at physiological levels appears to increase bone collagen synthesis by a direct effect on the osteoblast, whereas insulin at high concentrations has an additional action to increase the replication of collagen-synthesizing cells.

Animals↗
PubMed1984

Cortisol decreases bone formation by inhibiting periosteal cell proliferation.

We tested the hypothesis that the inhibitory effect of cortisol on bone growth is primarily on the periosteum. Fetal rat calvaria were maintained in organ culture, labeled with radioactive proline or thymidine, and then dissected to separate periosteum from the osteoblast-rich central bone. There was a dose- and time-dependent inhibition of thymidine incorporation into DNA in the periosteum which was significant at 24 h. These observations were further supported by decreases in the dry weight and DNA content of the periosteum at 96 h. Incorporation of thymidine and proline into the central bone were decreased only at 96 h. Pulse-chase studies using a high concentration of cortisol (10(-6) M) indicated that increased cell attrition may also play a role in the inhibitory effect of cortisol. We propose that the primary effect of cortisol on bone growth is an inhibition of proliferation of the periosteal cells which give rise to osteoblasts. The subsequent decrease in the incorporation of proline into the central bone may be the consequence of this inhibition.

Animals↗
PubMed1982

Regulation of collagen synthesis in fetal rat calvaria by 1,25-dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) inhibited type I collagen synthesis in the central bone but not the periosteum of fetal rat calvaria maintained in organ culture. The central bone synthesized primarily type I collagen, whereas the periosteum synthesized both types I and III collagen. Enhanced degradation of newly synthesized collagen seemed an unlikely mechanism for the observed decrease in collagen synthesis since 1,25-(OH)2D3 reduced both the labeling of intracellular procollagen and total [3H]hydroxyproline formation in fetal rat calvaria. As measured by cell-free translation of RNA extracted from calvaria, 1,25-(OH)2D3 decreased the level of functional procollagen mRNA and collagen synthesis to the same extent. Both collagen synthesis and procollagen mRNA levels were decreased as early as 3 h after exposure to 1,25-(OH)2D3 and were 50% of the control level by 24 hr. Upon removal of 1,25-(OH)2D3 from the cultures, collagen synthesis and procollagen mRNA remained depressed for 24 h but returned to control levels by 48 h. A reduction in collagen synthesis and procollagen mRNA was also observed in calvaria excised from 6-day-old rat pups given a single subcutaneous injection of 1,25-(OH)2D3 (1.6 ng/g body weight). We conclude that 1,25-(OH)2D3 inhibits the synthesis of type I collagen in the differentiated osteoblast by reducing the level of functional procollagen mRNA.

Animals↗
PubMed1982

Autoradiographic study of the effect of 1,25-dihydroxyvitamin D3 on bone matrix synthesis in vitamin D replete rats.

An autoradiographic technique using pulse labels of [3H]proline was developed to assess the early effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on bone matrix synthesis in vitamin D replete rats. Rats, 7 days old, were given 0.25 2.5, or 25 ng of 1,25(OH)2D3 for vehicle alone subcutaneously on days 1, 3, and 5 of the experiment. Rats received a subcutaneous injection of 100 microCi [3H]proline on days 2 and 6 and were killed on day 7. Calvaria and tibia were processed for autoradiography, and morphometric methods were developed to measure the rate and amount of bone matrix formed during the experimental period. When compared to control values, the amount and rate of formation of new bone matrix were both signifIcantly decreased in rats receiving 25 ng of 1,25(OH)2D3 and slightly, but not significantly, decreased in rats receiving 2.5 ng. We conclude that administration of pharmacologic doses of 1,25(OH)2D3 to vitamin D replete rat pups impairs the formation of collagenous bone matrix.

Animals↗
PubMed1982

Activation of serum complement inhibits collagen synthesis in fetal rat bone in organ culture.

Activation of rabbit serum complement caused a marked reduction in collagen synthesis but a much smaller change in noncollagen protein synthesis in fetal rat calvaria maintained in organ culture. In the periosteum of the fetal rat calvarium, both collagen and noncollagen protein synthesis were reduced, whereas in the central bone, presumably enriched in osteoblasts, only collagen synthesis was inhibited. This large decrease in bone collagen synthesis could not be attributed to enhanced degradation of newly synthesized collagen or its release into the culture medium. Activation of complement also stimulated the production of PGE in fetal rat calvaria. Antagonists of prostaglandin cyclooxygenase decreased prostaglandin synthesis but did not restore collagen synthesis in complement-treated bones, suggesting that complement decreases osteoblast collagen synthesis by a mechanism largely independent of prostaglandin production.

Animals↗
PubMed1981

Hormonal control of skeletal growth.

The endocrine control of skeletal growth involves not only calcium regulating hormones but also several systemic hormones and other factors. Many direct effects of these agents on skeletal growth have been demonstrated in vitro. While these direct effects may also predominate in vivo, and certainly are important in helping us understand how skeletal growth is controlled, it is likely that skeletal growth in vivo is determined by the complex interactions of a concert of hormones that play both permissive and regulatory roles. Additionally the effect of hormones may be modified by local or intrinsic responses of the skeleton or by changes in ion concentration. Phosphate may have a hormone-like function in the skeleton since high serum concentrations are associated with increased growth and low serum concentrations with impaired growth and mineralization (2, 5, 7, 37, 89). Because of these interactions, in vivo and in vitro studies on hormonal control of skeletal growth are often contradictory. A careful distinction between indirect and direct effects will help to interpret the information now available and to devise better studies in the future.

Animals↗
PubMed1980

Comparison of the effects of vitamin D metabolites on collagen synthesis and resportion of fetal rat bone in organ culture.

We compared the effects of four vitamin D metabolites, 1 alpha,25 dihydroxy vitamin D3 (1 alpha,25(OH)2D3), 1 alpha hydroxy vitamin D3 (1 alpha OH D3), 25 hydroxy vitamin D3 (25 OH D3), and 24R,25 dihydroxy vitamin D (24R,25(OH)sD3) on resorption and collagen synthesis in fetal rat bone maintained in organ culture. Resorption was quantitated by measuring the release of previously incorporated 45Ca from long bone shafts of 19-day fetal rats, and collagen synthesis was assessed by measuring the incorporation of 3H-proline into collagenase digestible protein (CDP) in calvaria from 21-day fetal rats. All four compounds stimulated bone resorption and inhibited collagen synthesis, but 1 alpha,25(OH)2D3 was approximately 1000 times more potent in both organ culture systems. Although the differences were small among the other three compounds, the order of potency was 1 alpha OH d3 > 25 OH D3 greater than or equal to 24R,25(OH)2D3. These results suggest that the receptor for 1 alpha 25(OH)2D3 in both bone resorbing and bone forming cells has similar affinities for several vitamin D metabolites.

Animals↗
PubMed1980

Parathyroid hormone alters collagen synthesis and procollagen mRNA levels in fetal rat calvaria.

Parathyroid hormone decreased the incorporation of [3H]proline into collagenase-digestible protein in cultured 21-day fetal rat calvaria but had little effect on the labeling of noncollagen protein. After 24 hr of culture, there was a 50% reduction in collaghen synthesis and a 40% decrease in the level of functional procollagen mRNA as measured in a reticulocyte lystate translation assay. The effect of parathyroid hormone on both parameters was detectable after 6 hr of treatment. In these cultures, there was also a substantial degradation or release of newly synthesized collagen from the calvaria, but parathyroid hormone had little effect on the release of collagen into the medium. These results suggest that parathyroid hormone inhibits collagen synthesis primarily by decreasing the steady-state level of procollagen mRNA.

Animals↗
PubMed1979

Prostaglandin synthesis by fetal rat bone in vitro: evidence for a role of prostacyclin.

Prostaglandin synthesis by fetal rat bones was examined by thin-layer chromatography of culture media after preincubation with labeled arachidonic acid. Cultures in rabbit complement (non-heat inactivated serum) were compared with cultures in heat-inactivated serum or cultures treated with indomethacin. The major complement-dependent products were PGE2, PGF2 alpha and 6-keto-PGF1 alpha, the metabolite of prostacyclin (PGI2). Since PGI2 had not been previously identified in bone its ability to stimulate bone resorption was tested. Repeated addition of PGI2 stimulated release of previously incorporated 45Ca from fetal rat long bones in both short-term and long-term cultures at concentrations of 10(-5) to 10(-9)M. Because of the short half life of PGI2 in solution at neutral pH, we tested a sulfur analog, thiaprostacyclin (S-PGI2) which was found to be a stimulator of bone resorption at concentrations of 10(-5) to 10(-6)M. These studies suggest that endogenous PGI2 production may play a role in bone metabolism. Since vessels produce PGI2 it is possible that PGI2 release may be responsible for the frequent association between vascular invasion and resorption of bone or calcified cartilage in physiologic remodeling and pathologic osteolysis.

Animals↗