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R N Rosier

Publications and source records attributed to R N Rosier.

18 recordsLinked to original sources
PubMed1992

Influence of prostaglandins on DNA and matrix synthesis in growth plate chondrocytes.

Prostaglandins are locally produced in a number of tissues in response to a variety of stimuli, including local growth factors and systemic hormones. The present investigation characterizes prostaglandin effects on growth plate chondrocytes. Since cyclic adenosine monophosphate (cAMP) may act as a prostaglandin-stimulated second messenger, the effects of prostaglandins A1, D2, E1, E2, F2 alpha, and I2 (10(-10)-10(-6) M) on cAMP levels and thymidine incorporation were evaluated. The stimulation of cAMP and thymidine incorporation by the various prostaglandin metabolites were dose dependent and highly correlated (r = 0.99, p less than 0.001). The magnitude of the effect varied but was maximal at 10(-6) M for each of the prostaglandins. Prostaglandins of the E series (E1 and E2) were the most potent, causing significant effects at 10(-10) M and with maximal 12- and 13-fold increases in DNA synthesis after a 24 h exposure. Prostaglandins D2 and A1 maximally stimulated thymidine incorporation by 4.7- and 3.1-fold but caused significant increases only at 10(-8) M. Prostaglandins F2 alpha and I2 were the least stimulatory, producing small but significant increases in thymidine incorporation at 10(-6) M (30 and 100% stimulations). A causal relationship between cAMP and thymidine incorporation was further verified by the ability of dibutyryl-cAMP to increase DNA synthesis. Long-term chondrocyte cultures treated continuously with PGE2 demonstrated an increase in cell number, confirming the proliferative effect. Indomethacin did not alter the potent dose-dependent stimulations of chondrocyte DNA synthesis by TGF-beta 1, basic FGF, or PTH, indicating that these known mitogens act independently of prostaglandin metabolism. PGE2 was further examined for its effects of matrix synthesis. PGE2 inhibited collagen synthesis with a maximal 42% decrease but did not alter noncollagen protein synthesis. In contrast, PGE2 maximally increased sulfate incorporation by 35% and caused a small dose-dependent inhibition in alkaline phosphatase activity. Thus, prostaglandins alter DNA and matrix synthesis in growth plate chondrocytes and may have an important role in chondrocyte metabolism in the growth plate, fracture callus, and other areas of endochondral ossification.

Alkaline Phosphatase↗
PubMed1992

Differential effects of parathyroid hormone on chick growth plate and articular chondrocytes.

Parathyroid hormone (PTH) binds specifically to the hypertrophic region of growth plate cartilage [16]. This specific binding suggests a role for this hormone in chondrocyte maturation. Enzymatically isolated chick articular and growth plate chondrocytes grown in monolayer culture were used to assay the direct effects of PTH on chondrocytes. The articular chondrocytes were unresponsive to PTH. The growth plate chondrocytes, however, demonstrated a marked mitogenic response to PTH, with a 39-fold increase of [3H]-thymidine incorporation into DNA. PTH also affected matrix production by the growth plate chondrocytes causing a twofold stimulation of proteoglycan synthesis as determined by the rate of 35SO4 incorporated into matrix macromolecules. Furthermore, PTH depressed collagen synthesis as measured by [3H]-proline incorporation. PTH caused a 12-fold increase in intracellular cAMP in growth plate chondrocytes but no increase in the articular cells. This specificity of PTH for growth plate chondrocytes suggests a possible regulatory role in enchondral ossification.

Alkaline Phosphatase↗
PubMed1992

Nonradioactive in situ hybridization using digoxigenin-labeled oligonucleotides. Applications to musculoskeletal tissues.

We have optimized a technique for in situ localization of specific mRNAs using digoxigenin-11-dUTP-labeled oligonucleotide probes. DNA probes were synthesized for type I and type II collagen as well as transforming growth factor-beta 1 and 2 (TGF beta 1 and TGF beta 2). Control experiments, such as competitive inhibition, nonsense sequence hybridization, and RNAse digestion all indicated that the technique was highly sensitive and specific. In sections of growth plate, type II collagen mRNA was predominantly expressed in the lower proliferative and upper hypertrophic zone, whereas chondrocytes in articular cartilage stained equally. These techniques then were applied to sections cut from archival pathology specimens of musculoskeletal tissues. Primitive chondrocytes in a chondrosarcoma expressed type I and type II collagen mRNA, but did not stain with the nonsense probe. Sections from an osteosarcoma, an aneurysmal bone cyst, and a neurofibroma also were investigated. The ability to use chemically synthesized oligonucleotide probes, the high resolution, and the short development times possible with this in situ procedure makes this technique appealing for applied research into the gene expression of normal and pathologic cellular events.

Animals↗
PubMed1991

Activation of osteoblast insulin-like growth factor-II/cation-independent mannose-6-phosphate receptors by specific phosphorylated sugars and antibodies induce insulin-like growth factor-II effects.

The phosphorylated monosaccharide, mannose-6-phosphate (M6P), causes a dose-dependent stimulation of alkaline phosphatase production by osteoblasts. The concentrations tested ranged from 0.1 to 30 mM. A maximal effect was reproducibly seen at 10-30 mM, and represented a 30% stimulation over control cells. Glucose-6-phosphate and fructose-1-phosphate also stimulated osteoblast alkaline phosphatase production, but not to the same extent as M6P. Sugar residues such as mannose, mannose-1-phosphate, and fructose-6-phosphate had no effect. The stimulatory effect of M6P is similar to that seen with insulin-like growth factor II(IGF-II). However, increasing doses of IGF-II did not further stimulate or add to the effect of 10 mM M6P. These data indicate that the mechanism for the transduction of the stimulatory signal may be similar for both IGF-II and M6P. They do not address, however, the possibility of separate or similar binding sites for the two agents. A specific polyclonal antibody to the IGF-II/cation-independent mannose-6-phosphate receptor (IGF-II/CI-MPR) elicits the same effects as M6P and IGF-II in these bone cells. Non-immune serum used as a control does not have any effect. These results suggest that activation of the osteoblast IGF-II/CI-MPR by either M6P or a specific antibody can evoke a biological response similar to that observed with IGF-II.

Alkaline Phosphatase↗
PubMed1991

Human prostatic acid phosphatase directly stimulates collagen synthesis and alkaline phosphatase content of isolated bone cells.

Human prostatic acid phosphatase (hPAP) directly enhances the differentiated characteristics of isolated bone cells in vitro. This enzyme, when added to cell cultures for 24 h in vitro stimulates collagen synthesis and the production of alkaline phosphatase. The effects are dose dependent, with statistically significant effects occurring from 0.1-100 nM hPAP. Concentrations higher than 100 nM do not evoke greater effects. The maximal effect of hPAP occurs between 12 and 24 h of exposure. The cells stimulated to the greatest degree are osteoprogenitor cells and osteoblasts. Fibroblasts isolated from the same tissue show a lesser sensitivity to hPAP. hPAP has no detectable effect on cell proliferation, as measured by radiolabeled thymidine incorporation or total DNA synthesis. None of the observations reported in this work can be attributed to contaminating proteins in the hPAP preparation. hPAP was radiolabeled with 125I and was used for affinity binding and cross-linking studies. Scatchard analysis of specific binding indicated the presence of 1.0 X 10(5) high affinity binding sites/cell, with a Kd of 6.5 nM. Cross-linking studies demonstrated the presence of one 320-kDa binding complex. The pH profile and kinetic determinations of Km and maximum velocity for hPAP were similar to those previously reported, except for the finding of positive cooperativity of the substrate with the enzyme under the conditions of our assay. We believe that the direct stimulation of bone-forming cells by hPAP may contribute to the sclerotic nature of skeletal bone around sites of neoplastic prostatic metastases and that the effect of the enzyme is probably mediated by a plasma membrane receptor.

Acid Phosphatase↗
PubMed1990

Synergistic effect of transforming growth factor beta and fibroblast growth factor on DNA synthesis in chick growth plate chondrocytes.

Transforming growth factor beta and fibroblast growth factor are mitogens for chick growth plate chondrocytes. TGF-beta stimulated a 3.5-fold increase, and FGF a 13.5-fold increase in the rate of thymidine incorporation after a 24 h exposure. TGF-beta and FGF were synergistic in chondrocytes, causing a 73-fold stimulation in thymidine incorporation compared with control. This synergistic response was not dependent upon the simultaneous presence of both mitogens. Sequential exposure of chondrocytes to TGF-beta and FGF in either order reproduced in large part the synergistic interaction observed when both growth factors were present simultaneously. The time required for induction of the subsequent synergistic response was brief and, in the case of TGF-beta, corresponded to the time required for [125I]TGF-beta receptor binding. EGF and PDGF were not mitogenic for chondrocytes, and neither of these factors enhanced the response of the cells to either TGF-beta or FGF. Finally, TGF-beta and FGF did not, either alone or in combination, elevate intracellular cAMP levels. These results emphasize the importance of examining growth factor effects in the context of other growth regulators. Furthermore, this specific and dramatic synergistic stimulation of thymidine incorporation may provide a useful tool in elucidating the mitogenic mechanism of the individual growth factors.

Animals↗
PubMed1990

Cytosolic free calcium concentrations in avian growth plate chondrocytes.

Isolated avian growth plate chondrocytes convert the acetoxymethyl ester (AM) form of Fura-2 quickly and efficiently to the Ca2(+)-sensitive pentacarboxylic acid (FA) form. Control experiments indicate that the Kd for intracellular Fura-2/FA is very close to that of extracellular Fura-2/FA at the same ionic strength and pH and that the Fura-2/FA fluorescence from indicator converted by intracellular organelles is quite small. Correcting for the effects of extracellular Fura-2/FA and partial hydrolysis products has improved the accuracy of determination of intracellular [Ca2+] over earlier measurements in chondrocytes. Cytosolic [Ca2+] in isolated growth plate chondrocytes (containing cells from each maturational stage) is found to require approximately 9 hours to recover from the isolation process. After this recovery period, cytosolic [Ca2+] in these cells converges to approximately 70 nM regardless of the [Ca2+] of the recovery medium, suggesting regulation of cytosolic [Ca2+] to a set point. Chondrocytes that are separated into maturationally distinct fractions using countercurrent centrifugal elutriation show an increase in cytosolic [Ca2+] with cellular maturation. The least mature resting cells have a [Ca2+] near 57 nM, while the most mature hypertrophic cells are around 95 nM.

Animals↗
PubMed1990

The production of transforming growth factor-beta by chick growth plate chondrocytes in short term monolayer culture.

Transforming growth factor-beta (TGF beta) is capable of regulating the proliferation and phenotypic expression of growth plate chondrocytes in culture. Chondrocytes were isolated from the growth plates from the long bones of 3- to 5-week-old chicks. Conditioned medium was harvested from short term monolayer cultures for the assay of TGF beta production by these cells. A receptor competition assay using [125I]TGF beta was used to quantitate the amount of TGF beta in the conditioned medium. Acid-activated conditioned medium contained 5.0 +/- 0.4 ng/ml TGF beta, while conditioned medium that had not been exposed to acid had undetectable levels of the peptide by this assay. The initial cell plating density was inversely related to the amount of TGF beta produced on a per cell basis. Growth plate chondrocytes separated by countercurrent centrifugal elutriation into maturationally distinct subpopulations had different rates of TGF beta production; hypertropic chondrocytes produced significantly more TGF beta (4.5 ng/10(6) cells) than the smallest chondrocytes isolated (2.3 ng/10(6) cells). A variety of other growth mediators were tested for their ability to influence TGF beta production by chondrocytes, and it was found that only basic fibroblast growth factor could significantly influence TGF beta production, producing a 6-fold increase in TGF beta recovered in the conditioned medium. The production of TGF beta by growth plate chondrocytes implicates it as an important autocrine or paracrine regulator in the process of endochondral calcification.

Animals↗
PubMed1990

Differential expression of biological effects in maturationally distinct subpopulations of growth plate chondrocytes.

Countercurrent centrifugal elutriation is an accurate and reproducible technique which can separate cells on the basis of size. This technique was utilized for the separation of isolated chick growth plate chondrocytes. Mean cellular volume, alkaline phosphatase activity, and Type X collagen synthesis progressively increased in each of seven successive separated fractions. The chondrocytes from the different fractions expressed a differential response to the growth regulator, transforming growth factor beta (TGF beta). TGF beta receptor binding to these cell fractions suggests that the different biological effects may be due to differences in the number of receptors expressed per cell as well as to differences in the proportion of high and low affinity TGF beta receptors present.

Alkaline Phosphatase↗
PubMed1989

Transforming growth factor beta: an autocrine regulator of chondrocytes.

Transforming growth factor beta (TGF-beta) is a ubiquitous regulator of cellular growth and differentiation. The present study investigated the effects of TGF-beta on chick growth plate chondrocyte proliferation, matrix synthesis, and alkaline phosphatase activity in short term cultures. TGF-beta markedly stimulated DNA synthesis in a dose-dependent manner, while collagen synthesis and cellular and matrix vesicle alkaline phosphatase activity were inhibited. Biologic effects of TGF-beta were correlated with binding to specific receptors, and both high and low affinity receptors were identified. Countercurrent centrifugal elutriation was used to fractionate growth plate chondrocytes to obtain populations of cells in different stages of maturation (effectively from different zones of the growth plate). TGF-beta showed increasing mitogenicity with increasing cellular maturation in the growth plate, with maximal stimulation in the proliferating and early hypertrophic cells. The smallest cells expressed only the high affinity receptor, while with hypertrophy there was increasing expression of the low affinity receptor and a progressive increase in the number of both receptors per cell. Furthermore, the dose-response curves for TGF-beta-stimulated DNA synthesis were not biphasic in the smaller cells, but became progressively more biphasic with cellular hypertrophy and expression of the low affinity receptor. Finally, TGF-beta activity was identified in partially purified chondrocyte conditioned medium by specific bioassay, indicating TGF-beta production by growth plate chondrocytes. The data suggests a potentially important autocrine function for TGF-beta in modulating chondrocyte proliferation and matrix synthesis in endochondral calcification.

Animals↗
PubMed1989

Pathologic bone formation.

The literature on pathologic bone formation is reviewed first on the basis of clinical syndromes and second in relation to newer knowledge of the origin of the bone-forming cells and regulatory molecules. Pathologic bone formation can be categorized into three groups based on the initiating stimulus: trauma, tumors, and idiopathic causes. In the trauma category, the formation of ectopic bone is concerned with major and minor traumatic incidents, surgery, burns, and other causes. In the tumor category, direct and reactive pathologic bone formation is discussed with an emphasis on the different neoplasms capable of ectopic bone formation. The category of idiopathic causes involves the formation of pathologic bone following neurologic injury and in systemic ossification disorders. The origin of the bone-forming cells in all forms of pathologic bone has not been unequivocally determined. However, there is evidence suggesting that these cells may arise from osteogenic stromal elements. Potent bone formation growth-regulating factors have been recently identified, and these molecules must also participate in the formation of pathologic bone. Increased understanding of the processes that control pathologic bone formation will lead to better methods of preventing and treating disorders of ossification.

Bone Marrow Cells↗
PubMed1989

Countercurrent centrifugal elutriation. High-resolution method for the separation of growth-plate chondrocytes.

Countercurrent centrifugal elutriation was used to separate, on the basis of size, isolated growth-plate chondrocytes in chicks. The mean cellular volume, activity of alkaline phosphatase, and synthesis of type-X collagen increased progressively in each of seven successive fractions. Slices of tissues that contained either proliferating or hypertrophic chondrocytes were also removed by manual dissection from the superficial and deep regions of the growth plate. These cells demonstrated differences in size and biochemistry that were similar to those observed in chondrocytes that were separated by elutriation. These differences included increased synthesis of proteoglycan and collagen in the larger chondrocytes. Radiolabeled hypertrophic chondrocytes were mixed with unlabeled resting and proliferating chondrocytes, and then were separated by elutriation. The radioactivity was recovered in fractions that contained the largest cells, confirming that differences in the sizes of the cells can be used to effect a zonal separation by centrifugal elutriation.

Alkaline Phosphatase↗
PubMed1988

Effect of transforming growth factor-beta on DNA synthesis by growth plate chondrocytes: modulation by factors present in serum.

Chondrocytes in the growth plate undergo rapid proliferation during the process of enchondral ossification. The factors that regulate this proliferative activity have not been defined although several local autocrine or paracrine growth factors have recently been discovered in cartilage. Transforming growth factor-beta 1 (TGF-beta) is an important regulator of cell metabolism and growth and is present in cartilage. The present study was designed to examine the influence of TGF-beta on DNA synthesis in chick epiphyseal chondrocytes. Chondrocytes were plated in serum-free (BSA-supplemented) medium or medium containing 10% FBS, and after 24 hours in monolayer culture, were treated with TGF-beta in identical medium. A 24 hour incubation with TGF-beta caused a biphasic dose-dependent increase in thymidine incorporation. The effect was greatly increased in serum-containing cultures where a maximal 20-fold increase in thymidine incorporation occurred compared with the 2 1/2-fold maximal increase obtained in serum-free cultures. The effect was present by 12 hours and maximal between 0.3 and 1.0 ng/ml of TGF-beta. Higher concentrations of TGF-beta were less stimulatory. The serum enhancement was dependent upon the simultaneous presence of TGF-beta and serum factors and was abolished when chondrocytes were plated and exposed to TGF-beta in medium containing dialyzed FBS (12-14 kD MW membrane). The nature of the uptake and incorporation of thymidine into DNA by individual chondrocytes appeared to be the same in both TGF-beta-treated and control cultures as the apparent Kms were similar. The present study indicates that TGF-beta increases DNA synthesis by growth plate chondrocytes. The effect is enhanced by factors present in serum.

Animals↗
PubMed1988

Effects of transforming growth factor-beta on matrix synthesis by chick growth plate chondrocytes.

Transforming growth factor-beta (TGF beta) is a local regulator of cell metabolism and growth. TGF beta increases the synthesis of collagen and enhances the deposition of matrix by almost all cells studied to date. The presence of TGF beta in cartilage suggests an important autocrine function, and the present study was designed to examine its influence on the matrix synthesis of chick epiphyseal chondrocytes. Chondrocytes were plated in serum-free (BSA-supplemented) medium or medium containing 10% fetal bovine serum (FBS), and after 24 h in monolayer culture were treated with TGF beta in identical medium. A 24-h incubation with TGF beta caused a dose-dependent decrease in collagen synthesis (-14%) and increase in noncollagen protein synthesis (+25%), with greater effects in serum-containing medium (-22% and +58%, respectively). Similarly, the stimulation of sulfate incorporation by TGF beta was greater in FBS-containing medium (+140%) than in serum-free medium (+70%). These changes were present by 6 h, were maximal in the 0.3-3.0 ng/ml dose range, and were found to reflect an alteration in extracellular protein synthesis. The enhancement of TGF beta effects by serum was abolished when chondrocytes were plated and exposed to TGF beta in medium containing dialyzed FBS (12-14K membrane). The present study indicates that TGF beta influences the synthesis of matrix components by growth plate chondrocytes. The effects are enhanced by factors present in serum.

Animals↗