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T L Chen

Publications and source records attributed to T L Chen.

At least 163 records · Page 9Linked to original sources
PubMed1985

Retinoic acid modulation of 1,25(OH)2 vitamin D3 receptors and bioresponse in bone cells: species differences between rat and mouse.

Retinoic acid (RA) caused a reduction in the level of 1,25(OH)2D3 receptors to 1/3 of control in rat osteoblast-like cells (ROB) while increasing the receptor level to 3-fold the control in mouse osteoblast-like cells (MOB). Scatchard analysis of receptor binding indicated that there was no change in affinity for 1,25(OH)2D3. The changes in receptor levels required time to develop and were dose-dependent. RA also modulated the ability of cells to respond to 1,25(OH)2D3 as measured by the induction of the enzyme 25(OH)D3-24 hydroxylase. Induction of enzyme activity by 1,25(OH)2D3 closely paralleled receptor level established by RA pretreatment. In MOB, the up-regulation of the receptor occurred despite the action of RA to inhibit DNA, RNA and protein synthesis. However, RA stimulation of 1,25(OH)2D3 receptor levels was blocked by the addition of cycloheximide or actinomycin D, indicating that the up-regulation required protein and RNA synthesis. The opposite effect of RA on mouse and rat cells suggests that important species-dependent factors modulate the action of retinoids on mammalian cells.

Animals↗
PubMed1985

A study of dynamic development in radiolesion and cell death of eukaryotes after exposure to ionizing radiation--the visualization of the dynamic reaction by means of cytochemical method with lactate dehydrogenase (LDH) as a nuclear marker and radiation-induced nuclear anomalization by an ultramicrostructural manifestation: a dynamic explanation.

The present work deals with the radiation effect on adult rats suffering from acute, whole-body irradiation with cobalt-60 gamma rays of dosages 100R and 300R respectively. The morphological alteration of the lymphocytic nucleus and exhibition of cytochemical reaction of LDH in intra- or extra-nuclear region has been observed at the end of predetermined time intervals after irradiation. A close relationship between the percentage of damage of the lymphocytic nucleus and that of positivity of LDH reaction is seen from their variations with time parallel. From technical color microphotographs which exhibit cytochemical reactions, the existence of enzyme LDH in the nucleus so far obscure has been visually recognized. The events happening in enzymic reaction and the leakage of enzyme across the nuclear envelope reflect the dynamic nature. The ultrastructural alteration of nuclear anomalization confirms the dynamic development of inducing radiolesion. The experimental facts inspire us to attempt an explanation of cellular radiolesion and cell death owing to ionization radiation in light of the dynamic concept.

Animals↗
PubMed1984

Modulation of PTH-stimulated cyclic AMP in cultured rodent bone cells: the effects of 1,25(OH)2 vitamin D3 and its interaction with glucocorticoids.

Parathyroid hormone (PTH)-stimulated cyclic adenosine monophosphate (cAMP) in rat osteoblastlike (OB) cells has been shown to be modulated by steroid hormones; glucocorticoids are known to increase the level, while the effects of 1,25(OH)2D3 are inhibitory. In the present study, we found that the PTH-stimulated cAMP responses are similar in neonatal mouse and fetal rat OB cells. Dexamethasone (0.13-13 nM) augmented PTH-stimulated cAMP in both species. Mouse cells showed a higher maximal response to dexamethasone (100% increment) than rat cells (60-70% increment) with similar sensitivity to dexamethasone (ED50 approximately 1.0 nm). On the other hand, 1,25(OH)2D3 decreased PTH-stimulated cAMP, but the effect required pharmacological levels of hormone; mouse cells responded at a lower dose (1.3 nM) and were more sensitive than rat cells (responded at 13 nM) to 1,25(OH)2D3 treatment. Introduction of physiological concentrations of 1,25(OH)2D3 (0.013-1.3 nm) in addition to dexamethasone (13 nM) resulted in a synergistic enhancement of PTH-stimulated cAMP in rat cells. In contrast, a dose-dependent antagonistic effect was observed in mouse cells. In summary, our findings demonstrate species and concentration-dependent differences in hormonal responses to 1,25(OH)2D3 and a complex interplay among PTH, dexamethasone, and 1,25(OH)2D3.

Animals↗
PubMed1984

1,25-dihydroxyvitamin D resistance, rickets, and alopecia: analysis of receptors and bioresponse in cultured fibroblasts from patients and parents.

To investigate further the cellular defects of vitamin D-dependent rickets type II with alopecia, we studied 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors and the response to 1,25-(OH)2D3 in cultured skin fibroblasts from rachitic patients. Our studies included cells from four affected patients from three kindreds and their parents and cells from five normal subjects. We measured total 1,25-(OH)2D3 receptor binding in cell extracts and the capacity of 1,25-(OH)2D3 to induce the enzyme 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) as a marker of functional response. In normal fibroblasts, the 1,25-(OH)2D3 maximal binding capacity was 52 +/- 5 fmol/100 micrograms DNA (mean +/- SE), and the apparent dissociation constant (Kd) was 0.05 +/- 0.01 nM. The maximal induced 24-hydroxylase activity after 1,25-(OH)2D3 treatment was 11.5 +/- 1 fmol/10(6) cells X 30 min, and the dose of 1,25-(OH)2D3 that achieved half-maximal induction was 2.3 +/- 0.3 nM. Fibroblasts from all four rachitic patients had the same defect: no measurable 1,25-(OH)2D3 receptor binding and no detectable response above basal activity even after high doses of 1,25-(OH)2D3. Cells from all parents except one had normal 1,25-(OH)2D3 binding characteristics and normal 24-hydroxylase bioresponse to 1,25-(OH)2D3. One parent despite a normal phenotype had only half the normal level of binding sites and only half the normal bioresponse. In summary, the cultured fibroblasts from four affected children representing three different kindreds with 1,25-(OH)2D3 resistance failed to exhibit detectable 1,25-(OH)2D3 receptors. We postulate that this biochemical defect produced both the inability to respond to 1,25-(OH)2D3 in vitro and the 1,25-(OH)2D3 resistance in vivo. The obligate heterozygotic parents were normal, except for one who had both half the normal number of receptors and half the normal response to 1,25-(OH)2D3. The data confirm the critical role of the receptor in 1,25-(OH)2D3 action and the close coupling of receptor content and functional responsiveness.

Alopecia↗
PubMed1983

1 alpha,25-dihydroxyvitamin D3 receptors in cultured rat osteoblast-like cells. Glucocorticoid treatment increases receptor content.

The direct actions of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) in target cells are initiated by the binding of hormone to specific receptor sites. The aims of this study were to characterize the receptor for 1,25(OH)2D3 in cultured rat osteoblast-like (OB) cells and to determine whether these receptors are regulated by either the culture cycle itself or glucocorticoid treatment. The 1,25(OH)2D3 receptor in rat OB cells exhibited the same apparent binding affinity (Kd = 0.1 nM) and sedimentation coefficient (3.2 S) as mouse OB cells and receptors in other target organs. However, the receptor concentration in rat OB cells was substantially lower than mouse OB cells (approximately 20%). The concentration of receptors in rat OB cells did not show a correlation with the rate of DNA synthesis and therefore did not exhibit an endogenous rhythm in receptor concentration as was previously seen in mouse OB cells. Also in contrast to mouse cells, where glucocorticoids caused a decrease in receptor level, dexamethasone induced a marked increase in receptor binding throughout the culture cycle. This increase was due to an increase in receptor number with no change in receptor affinity. The change was glucocorticoid-specific, dose-dependent with half-maximal stimulation occurring between 1.3 and 13 nM and exhibited a latent period of at least 4 h. The independence of receptor concentration from DNA synthesis rate was established by assessing receptors after stimulating cell proliferation with epidermal growth factor and inhibiting it with hydroxyurea. Neither treatment altered basal 1,25(OH)2D3 receptor concentration or prevented the marked increase in receptor levels elicited by dexamethasone. We conclude that although the biochemical characteristics of 1,25(OH)2D3 receptors are indistinguishable in rat and mouse OB cells, there are genuine species differences in the regulation of the receptor number as it relates to DNA synthesis rate and response to glucocorticoids.

Animals↗
PubMed1983

Effects of 1 alpha,25-dihydroxyvitamin D3 and glucocorticoids on the growth of rat and mouse osteoblast-like bone cells.

The effects of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its interaction with glucocorticoids to regulate bone cell growth were studied in osteoblast-like (OH) cell cultures. Owing to our earlier findings that species difference and cell density at the time of treatment modified hormonal responses, comparisons were made between rat and mouse cells and sparse and dense cultures. 1,25(OH)2D3 inhibited cell proliferation in both species regardless of cell density. The magnitude of inhibition was larger in mouse cells, but the sensitivity to 1,25(OH)2D3 was the same for both species. Other metabolites, 25(OH)D3 and 24R,25(OH)2D3, were greater than 100-fold less potent than 1,25(OH)2D3 even in serum-free medium, which is similar to their ratio of affinity for the 1,25(OH)2D3 receptor. Dexamethasone, as previously shown, inhibited sparse and dense mouse cell cultures and sparse rat cell cultures while stimulating dense rat cell cultures to grow. The inhibitory actions of 1,25(OH)2D3 were not additive to the inhibitory dexamethasone effects. However, 1,25(OH)2D3 addition resulted in attenuation of the stimulatory effect of dexamethasone. These responses to 1,25(OH)2D3 and dexamethasone were dependent on cell density and not selective attachment of certain cell types at either plating density. In conclusion, the findings demonstrated that 1,25(OH)2D3 exerts an inhibiting action on both mouse and rat bone cell proliferation. This effect must be reconciled with the in vivo beneficial actions of 1,25(OH)2D3 on bone metabolism. Also, the likelihood of decreased cell number must be considered when biochemical activities are assessed after vitamin D treatment in vitro.

Animals↗
PubMed1983

Glucocorticoid modulation of cell proliferation in cultured osteoblast-like bone cells: differences between rat and mouse.

Glucocorticoids are important regulators of cell proliferation, but divergent effects have been noted in various systems. In this study, the actions of glucocorticoids on cell proliferation were examined in primary cultures of osteoblast-like cells from perinatal rodents. The aim of the study was to determine whether rat and mouse bone cells exhibit species differences in their responses to glucocorticoids and whether the response of either species was influenced by the cell density and time of treatment. Cultured osteoblast-like cells were treated with dexamethasone or ethanol vehicle (less than 0.05%). Changes in the proliferation rate were assessed by measurement of cell number, DNA content, and thymidine incorporation rate. We found that the response to dexamethasone was dependent on the cell density at the time of treatment in rat cultures: inhibitory in sparse and stimulatory in dense cultures. In contrast, dexamethasone inhibited mouse cell proliferation at all cell densities. The stimulatory effects in rat cells were seen after 4 days of treatment but not after 2 days. Both the stimulatory and inhibitory effects in rat and mouse cells occurred between dexamethasone concentrations of 1.3-13 nM, with half-maximal effects seen at 6 nM. The divergent responses in sparse and dense rat cultures were found not to result from the selective attachment of cell subpopulations at different plating densities. Epidermal growth factor was mitogenic in both species. The effects of dexamethasone were not modified by the presence of epidermal growth factor in either low (1%) or high (10%) serum concentrations. In conclusion, these findings reemphasize the importance of species differences and the difficulty in comparing one system to another, even with closely related species such as mouse and rat. Furthermore, the data show that the culture conditions at the time of treatment dramatically influence the responses of these cells to glucocorticoids. These factors must be considered in studies of the hormonal regulation of bone cell proliferation.

Animals↗
PubMed1980

Demonstration of glucocorticoid receptors in the adrenal cortex: evidence for a direct dexamethasone suppressive effect on the rat adrenal gland.

The adrenal cortex was evaluated for the presence of glucocorticoid receptors and functions. Substantial binding of [3H]dexamethasone was observed in aminoglutethimide-treated, hypophysectomized, and intact rats. Further studies demonstrated binding in cultured bovine adrenocortical cells and in Y-1 cells, a cloned murine cell line of adrenal cortical origin. Scatchard analysis of specific binding data in cytosol from hypophysectomized rats revealed an apparent Kd of approximately 15 nM and a receptor content (Nmax) of 123 fmol/mg cytosol protein. Analysis of Y-1 cell cytosol showed a Kd of approximately 17 nM and Nmax of 190 fmol/mg protein. The binding site in hypophysectomized rats had the following steroid specificities: high affinity for dexamethasone, corticosterone, and progesterone; moderate affinity for 11 beta-cortisol, and low affinity for testosterone, estradiol, pregnenolone, and 11 alpha-cortisol. Sedimentation in sucrose density gradients revealed 8S binding peaks in cytosols prepared from intact rat adrenal glands, Y-1 cells, and cultured bovine adrenocortical cells. Time- and temperature-dependent nuclear uptake of [3H]dexamethasone in Y-1 cells was demonstrated. In vivo treatment of hypophysectomized rats with dexamethasone significantly enhanced the rate of adrenal atrophy. ACTH stimulation tests in hypophysectomized rats showed a decreased corticosterone response in dexamethasone-treated rats compared to that in control animals. However, in vitro, there was no evidence for an effect of dexamethasone on ACTH-stimulated corticosterone production. The data indicate that the adrenal cortex possesses a high affinity binding site that fulfills the criteria for a glucocorticoid receptor. Glucocorticoid administration enhances adrenal atrophy and impairs adrenal function. We speculate that this action contributes to the suppressive effect of glucocorticoids on the pituitary-adrenal axis.

Adrenal Cortex↗
PubMed1979

A receptor-like binding macromolecule for 1 alpha, 25-dihydroxycholecalciferol in cultured mouse bone cells.

1alpha, 25-Dihydroxycholecalciferol (1,25-(OH)2D3), the active form of vitamin D, like other steroid hormones, initiates its action by binding to cytoplasmic receptors in target cells. Although the 1,25-(OH)2D3 receptor has been well studied in intestine, little information beyond sucrose gradient analyses is presently available from mammalian bone. We, therefore, employed primary cultures of mouse calvarial cells to characterize the mammalian receptor in bone. A hypertonic molybdate-containing buffer was found to protect receptor binding. On hypertonic sucrose gradients, the 1,25-(OH)2-[3H]D3 binder sedimented at 3.2 S. Scatchard analysis of specific 1,25-(OH)2[3H]D3 binding sites at 0 degrees C yielded an apparent Kd of 0.26 nM and an Nmax of 75 fmol/mg of cytosol protein. Competitive binding experiments revealed the receptor to prefer 1,25-(OH)2D3 greater than 25-(OH)-D3 = 1 alpha-(OH)-D3 greater than 24R,25-(OH)2D3; vitamin D3, dihydrotachysterol, sex steroids, and glucocorticoids exhibited negligible binding. As shown in other systems, the receptor could be distinguished from a 25-(OH)-[3H]D3 binder which sedimented at approximately 6 S. In summary, cultured mouse calvarial cells possess a macromolecule with receptor-like properties. This system appears to be an ideal model for the investigation of 1,25-(OH)2D3 receptor binding and action in mammalian bone.

Animals↗
PubMed1979

Glucocorticoid receptors and actions in subpopulations of cultured rat bone cells. Mechanism of dexamethasone potentiation of parathyroid hormone-stimulated cyclic AMP production.

We have previously shown that bone cells possess glucocorticoid receptors and that, in addition to being inhibitory to cell growth, glucocorticoid treatment potentiates the ability of parathyroid hormone (PTH) to stimulate cyclic AMP (cAMP) formation. This study extends those observations to specific subpopulations of bone cells and explores the mechanism of the cAMP augmentation. Subpopulations of cultured bone cells derived from 20-d-old fetal rat calvaria were enriched for "osteoblast-like" (OB) and "osteoclast-like" (OC) cells by sequential collagenase digestion. OC cells released during the first 30 min of collagenase digestion were characterized by low alkaline phosphatase activity, a cAMP response to salmon calcitonin (CT), but only a small cAMP response to bovine PTH. In contrast, OB cells released between 30 and 120 min of collagenase digestion, possessed high alkaline phosphatase activity, responded with a large cAMP rise to PTH, but exhibited no response to CT. Glucocorticoid receptors, with similar properties, were demonstrated in both populations (K(d) congruent with 5 nM, N(maximum) congruent with 400 fmol/mg cytosol protein). Dexamethasone equivalently inhibited cell growth and alkaline phosphatase activity in both populations. Dexamethasone potentiation of cAMP generation occurred after PTH but not CT stimulation. A greater enhancement of cAMP generation observed in OB cells appears to result from two glucocorticoid actions: (a) stimulation of adenylate cyclase and (b) inhibition of phosphodiesterase. Only the latter mechanism was found in OC cells. Dexamethasone-treated cells showed an increase in both sensitivity and maximal response of cAMP to PTH. The possible relationship of these actions to the mechanism of glucocorticoid-induced osteopenia is discussed.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1978

Fetal rat bone in organ culture: effect of bone growth and bone atrophy on the comparative losses of 45Ca and 3H-tetracycline.

Fetal rat bones were cultured in either growth-inducing or resorption-inducing media to study mineral losses during bone growth and atrophy in vitro. Whole radii and ulnae from 19-day-old fetal rats, prelabeled with 45Ca and/or 3H-tetracycline, were cultured intact or cut, and then digested by collagenase to obtain the calcified portion of the bones. Three- to five-fold more 3H-tetracycline than 45Ca was lost from the calcified portion when the bones were cultured for 4 days in growth-inducing media. Similar small amounts of 45Ca were lost from live and killed bones, but more 3H-tetracycline was lost from live bones than from killed bones. More 3H-tetracycline was released into the growth medium with a low concentration of calcium (0.5 mM) than when the calcium concentration was high (1.0 mM); no significant difference was seen in the release of 45Ca into the medium at different calcium concentrations. Larger amounts of both isotopes were lost when the prelabeled bones were cultured in resorption-inducing media than in growth-inducing media. When parathyroid hormone stimulated bone resorption in a resorption-inducing medium, equal proportions of both isotopes and bone collagen were lost. Greater losses of 3H-tetracycline than of 45Ca suggest that 45Ca was conserved locally during the resorption that accompanies bone growth, but not during resorption that accompanies bone atrophy.

Animals↗