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Biomedical subjects

T Bellido

Publications and source records attributed to T Bellido.

32 records · Page 2Linked to original sources
PubMed1995

New insights into the cellular, biochemical, and molecular basis of postmenopausal and senile osteoporosis: roles of IL-6 and gp130.

It is well established that osteoclasts, the cells responsible for bone resorption, are derived from hematopoietic progenitors (CFU-GM), whereas the bone-forming osteoblasts are of the same lineage as the mesenchymal stromal cells of the bone marrow. Moreover, it is widely accepted that osteoclast formation depends on cells of the stromal/osteoblastic lineage. The appreciation of the ontogeny of osteoclasts and osteoblasts, the interaction between them, and the role of local factors that regulate their development has led to the emergence of new insights into the pathophysiology of the osteopenias associated with estrogen deficiency and senescence. Consistent with histomorphometric data from humans, there is now evidence from studies in animal models suggesting that a critical cellular change caused by the loss of ovarian, as well as testicular, function is an increase in osteoclastogenesis. This change is apparently mediated by an increase in the production of the osteoclastogenic cytokine interleukin-6 by cells of the bone marrow, which follows the removal of an inhibiting control of estrogens or androgens on IL-6. The inhibiting effect of sex steroids on IL-6 production is mediated by their respective receptors and is exerted indirectly on the transcriptional activity of the proximal 225 bp sequence of the IL-6 gene promoter. Besides its effects on IL-6 production, loss of gonadal function may also cause an increase in the sensitivity of the osteoclastic precursors to the action of cytokines such as IL-6, due to an upregulation of the gp130 signal transduction pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Regulation of interleukin-6, osteoclastogenesis, and bone mass by androgens. The role of the androgen receptor.

Interleukin-6 is an essential mediator of the bone loss caused by loss of estrogens. Because loss of androgens also causes bone loss, we have examined whether the IL-6 gene is regulated by androgens, and whether IL-6 plays a role in the bone loss caused by androgen deficiency. Both testosterone and dihydrotestosterone inhibited IL-6 production by murine bone marrow-derived stromal cells. In addition, testosterone, dihydrotestosterone, and adrenal androgens inhibited the expression of a chloramphenicol acetyl transferase reporter plasmid driven by the human IL-6 promoter in HeLa cells cotransfected with an androgen receptor expression plasmid; however, these steroids were ineffective when the cells were cotransfected with an estrogen receptor expression plasmid. In accordance with the in vitro findings, orchidectomy in mice caused an increase in the replication of osteoclast progenitors in the bone marrow which could be prevented by androgen replacement or administration of an IL-6 neutralizing antibody. Moreover, bone histomorphometric analysis of trabecular bone revealed that, in contrast to IL-6 sufficient mice which exhibited increased osteoclast numbers and bone loss following orchidectomy, IL-6 deficient mice (generated by targeted gene disruption) did not. This evidence demonstrates that male sex steroids, acting through the androgen-specific receptor, inhibit the expression of the IL-6 gene; and that IL-6 mediates the upregulation of osteoclastogenesis and therefore the bone loss caused by androgen deficiency, as it does in estrogen deficiency.

Animals↗
PubMed1994

Modulation of calcium uptake in cultured cardiac muscle cells by 1,25-dihydroxyvitamin D3.

Recent studies have provided evidence indicating that 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates calcium influx through Ca2+ channels in isolated chick heart tissue and cells. Studies were performed both to evaluate the characteristics of the hormone action in cultured myocytes and to obtain information on the metabolic pathway which mediates its effects. Treatment of 70-80% confluent chick embryo myocyte monolayers with 1,25(OH)2D3 induced a fast (within 3-5 min) stimulation of 45Ca uptake which was dose-dependent, maximum responses (130% above controls) being elicited at a concentration of 10(-10) M. Physiological levels of 25(OH)D3 and 24,25(OH)2D3, and the synthetic analog 1 alpha (OH)D3, had lower activity. Coincident with the rapid changes in Ca uptake, 1,25(OH)2D3 significantly increased cAMP levels. The hormone-induced increase in cAMP was not blocked by nifedipine. Compound SQ 22536, a specific inhibitor of adenylate cyclase, completely suppressed the effects of the sterol on cAMP and Ca uptake. Furthermore, GDP-beta-S inhibited the increase in Ca uptake by 1,25(OH)2D3. These results involve the adenylate cyclase pathway and the participation of G proteins in 1,25(OH)2D3 stimulation of Ca influx in chick heart cells.

Animals↗
PubMed1994

17 beta-Estradiol inhibits expression of human interleukin-6 promoter-reporter constructs by a receptor-dependent mechanism.

We previously reported that 17 beta-estradiol inhibits cytokine-stimulated bioassayable IL-6 and the steady-state level of IL-6 mRNA. To determine the molecular basis of this effect, the transient expression of chloramphenicol acetyltransferase (CAT) reporter plasmid driven by the human IL-6 promoter was studied here in HeLa or murine bone marrow stromal cells (MBA 13.2). 17 beta-estradiol (10(-8) M) completely suppressed stimulated CAT expression in HeLa cells cotransfected with IL-6/CAT constructs and a human estrogen receptor (hER) expression plasmid; but had no effect on reporter expression in HeLa cells not transfected with hER. 17 beta-estradiol also inhibited stimulated expression in MBA 13.2 cells (which express the estrogen receptor constitutively) without the requirement of cotransfection of the hER plasmid. The hormonal effects were indistinguishable between constructs containing a 1.2-kb fragment of the 5' flanking region of the IL-6 gene or only the proximal 225-bp fragment. However, yeast-derived recombinant hER did not bind to the 225-bp segment in DNA band shift assays, nor did the 225-bp fragment compete for binding of an estrogen response element oligonucleotide to yeast-derived estrogen receptor. These data suggest that 17 beta-estradiol inhibits the stimulated expression of the human IL-6 gene through an estrogen receptor mediated indirect effect on the transcriptional activity of the proximal 225-bp sequence of the promoter.

Base Sequence↗
PubMed1994

Vitamin D and the hematolymphopoietic tissue: a 1994 update.

Monocytes/macrophages and activated (but not resting) lymphocytes as well as certain subsets of thymocytes express the VDR. This protein is indistinguishable from the classical 50-kDa VDR and is encoded by an mRNA with identical nucleotide sequence to that of the human intestinal VDR. Acting via the VDR, 1,25(OH)2D3 modulates the production of a plethora of monocyte, lymphocyte, and bone marrow stromal cell products, including several interleukins and other cytokines, as well as various oncogenes and transcription factors. However, these hormonal effects vary depending on the signals used to activate blood mononuclear cells; moreover, each of the effects of the hormone can be either attenuated, abolished, or even reversed from negative to positive in the presence of phorbol esters. Lymphocytes also express a previously unrecognized 80-kDa cytosolic protein that shares immunologic cross-reactivity with the VDR. This protein is induced on activation and is downregulated by 1,25(OH)2D3, whereas the VDR is upregulated by 1,25(OH)2D3. In contrast to the signal-dependent effects of the hormone on cytokine production and lymphocyte proliferation, the effects of 1,25(OH)2D3 on the 80-kDa protein and VDR are independent of the activation signals. This apparent mechanistic distinction raises the possibility that the signal-independent effects of 1,25(OH)2D3 on the 80-kDa protein and the VDR might be due to direct interactions of the 1,25(OH)2D3-VDR complex with specific response elements (negative and positive VDREs, respectively) on these two genes; as opposed to the signal-dependent effects that might be due to influences of the 1,25(OH)2D3-VDR complex on other transcription factors that are generated in response to the different activation stimuli. Consistent with the second part of this contention, we have recently found that 1,25(OH)2D3 regulates the 50-kDa DNA binding subunit of the pleiotropic transcription factor NF-kappa B and the 105-kDa precursor of this subunit; as well as other members of the rel-related family of proteins, including v-rel and its normal cellular homolog c-rel, in activated normal human lymphocytes. Besides its influence on immune cell products, 1,25(OH)2D3 is a potent agent for the differentiation of cells of the myeloid lineage. In addition, 1,25(OH)2D3 stimulates the fusion and differentiation of hematopoietic progenitors into osteoclasts, an effect which accounts for the potent role of the hormone in bone resorption.(ABSTRACT TRUNCATED AT 400 WORDS)

Adjuvants, Immunologic↗
PubMed1993

Evidence for the participation of protein kinase C and 3',5'-cyclic AMP-dependent protein kinase in the stimulation of muscle cell proliferation by 1,25-dihydroxy-vitamin D3.

Treatment with 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) (1-12 h, 10(-10) M) stimulates DNA synthesis in proliferating myoblasts, with an early response at 2-4 h of treatment followed by a maximal effect at 10 h. To investigate the mechanism involved in the mitogenic action of the hormone we studied the possible activation of intracellular messengers by 1,25(OH)2D3. The initial phase of stimulation of [3H]thymidine incorporation into DNA by the sterol was mimicked by the protein kinase C activator tetradecanoylphorbol acetate (TPA) in a manner which was dose dependent and specific as the inactive analog 4 alpha-phorbol was without effect. Maximal responses to TPA (100 nM) were obtained at 4 h. Staurosporine, a protein kinase C inhibitor, blocked the effect of 1,25(OH)2D3 on myoblast proliferation at 4 h. In addition, a fast (1-5 min) elevation of diacylglycerol levels and membrane-associated protein kinase C activity was observed in response to 1,25(OH)2D3. The adenylate cyclase activator forskolin (20 microM) and dibutyryl-cAMP (50 microM) increased DNA synthesis reproducing the second 1,25(OH)2D3-dependent stimulatory phase at 10 h. Inhibitors of protein kinase A blocked the increase in muscle cell DNA synthesis induced by 1,25(OH)2D3 at 10 h. Significant increases in cyclic AMP levels were detected in myoblasts treated with the sterol for 1-10 h. The calcium channel antagonist nifedipine (5-10 microM) abolished both the effects of 4-h treatment with 1,25(OH)2D3 or TPA and 10-h treatment with 1,25(OH)2D3 or dibutyryl-cAMP. Similar to the calcium channel agonist Bay K8644, 1,25(OH)2D3 stimulated myoblast 45Ca uptake and its effects were blocked by nifedipine.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

Demonstration of estrogen and vitamin D receptors in bone marrow-derived stromal cells: up-regulation of the estrogen receptor by 1,25-dihydroxyvitamin-D3.

We have shown earlier that 17 beta-estradiol inhibits cytokine-induced interleukin-6 (IL-6) production by bone marrow-derived stromal cells as well as osteoblasts, two types of cells with a critical influence on osteoclast development, and that ovariectomy causes an IL-6-mediated up-regulation of osteoclastogenesis in mice. Prompted by this, we have searched here for the presence of estrogen receptors (ERs) in two murine bone marrow-derived stromal cell lines, +/+ LDA11 and MBA 13.2, and the osteoblast-like cell line MC3T3-E1. All three cell lines exhibited high affinity saturable binding for [125I]17 beta-estradiol with a dissociation constant of approximately 10(-10) M and concentration of binding sites of 260 +/- 30, 170 +/- 10, and 90 +/- 10 sites per cell, respectively. In addition, we amplified complementary DNA from the stromal cell lines by polymerase chain reaction using oligonucleotide primers flanking the DNA binding domain of the murine uterine ER. The amplified product showed an identical nucleotide sequence to the DNA binding domain of the murine uterine receptor. Consistent with the functionality of the ER in stromal cells, and specifically its role in the regulation of IL-6 by 17 beta-estradiol, we found that the pure estrogen antagonist ICI 164,384 completely prevented the effect of 17 beta-estradiol on IL-6. All three cell lines also expressed receptors for 1,25-dihydroxyvitamin-D3 [1,25(OH)2D3] (dissociation constant, approximately 10(-10) M), with a concentration of binding sites of 490 +/- 20, 920 +/- 20, and 1110 +/- 70 sites per cell, respectively. 1,25(OH)2D3 treatment of the stromal cells caused a 2-fold increase in the concentration of ERs and a decrease in cell proliferation. These data establish that bone marrow-derived stromal cells express functional estrogen as well as vitamin D receptors, which serve to mediate actions of their respective ligands on the biosynthetic activity of these cells and presumably the effects of these two steroid hormones on osteoclastogenesis.

Animals↗
PubMed1991

Effects of 1,25-dihydroxy-vitamin D3 on phosphate accumulation by myoblasts.

The effects of 1,25-dihydroxy-Vitamin D3 on phosphate uptake by cultured chick embryonic muscle cells were investigated. Preincubation of primary myoblast cultures during 4-24 hours with physiological levels of 1,25(OH)2D3 resulted in a significant stimulation of velocity and total capacity of phosphate accumulation by the cells. Maximal responses were obtained at 8 hours of treatment with the sterol. In agreement with previous studies, 25-hydroxy-Vitamin D3 also stimulated myoblast phosphate uptake. 24,25-dihydroxy-Vitamin D3 and vitamin D3 were ineffective. Evidence was obtained indicating that 1,25(OH)2D3 affects the Na(+)-linked component of muscle cell phosphate uptake through a mechanism dependent on "de movo" protein and RNA synthesis.

Animals↗
PubMed1987

The phospholipid and fatty acid composition of skeletal muscle cells during culture in the presence of vitamin D-3 metabolites.

The phospholipid and fatty acid composition of primary cultures (24 h) of chick embryo skeletal muscle myoblasts treated for 4-24 h with physiological concentrations of 1,25-dihydroxyvitamin D-3 and 25-hydroxyvitamin D-3 were analyzed. 25-Hydroxyvitamin D-3 did not alter the relative amounts of individual muscle cell phospholipids whereas 1,25-dihydroxyvitamin D-3 significantly increased phosphatidylcholine content, mainly at the expense of a decrease in phosphatidylethanolamine concentration. The increase in phosphatidylcholine occurred at a faster rate during the first 8 h than in the subsequent 8-24 h treatment period. A similar time course in 1,25-dihydroxyvitamin D3-dependent changes in myoblast calcium uptake has been observe. In addition, this metabolite markedly increased (100%) the arachidonate content of myoblast phosphatidylcholine near the fusion stage of the cells (24 h of treatment). The levels of docosahexaenoate, a minor polyunsaturated fatty acid, in phosphatidylcholine and phosphatidylethanolamine were also substantially elevated by 1,25-dihydroxyvitamin D-3. No significant changes in fatty acid composition in response to 25-hydroxyvitamin D-3 were observed. Modifications in phospholipids and polyunsaturated fatty acids may play a role in the effects of 1,25-dihydroxyvitamin D-3 on muscle cell calcium transport and differentiation.

Animals↗
PubMed1987

Phosphate accumulation by muscle in vitro and the influence of vitamin D3 metabolites.

Phosphate accumulation by muscle in vitro and the effects of vitamin D3 metabolites thereupon were studied in cultures of chick embryo skeletal muscle myoblasts and intact chick soleus muscles. A significant proportion of phosphate accumulation by the cells was Na+-dependent, saturable with respect to phosphate, energy-dependent and inhibited by ouabain and arsenate, in agreement with the operation of a Na+-phosphate cotransport system in the muscle cell plasma membrane as has been described for intestine and kidney. This was further supported by the demonstration of substrate-saturable phosphate uptake in sarcolemma vesicles isolated from chick skeletal muscle. Preincubation of myoblast and soleus muscle cultures with physiological levels of 25-hydroxy-vitamin D3 resulted in a significant stimulation of phosphate accumulation by cultures. 1,25-dihydroxy-vitamin D3 had no effects on the differentiated tissue whereas it markedly increased phosphate accumulation by embryonic muscle cells. In addition, it could be shown that 25-hydroxy-vitamin D3 affects the Na+-linked component of cell phosphate uptake through a mechanism dependent on de novo protein synthesis.

24,25-Dihydroxyvitamin D 3↗
PubMedDate not supplied

Stimulation of myoblast membrane protein synthesis by 25-hydroxy-vitamin D3.

The effects of 25-hydroxy-vitamin D3 (25 OHD3) on myoblast protein synthesis were studied in connection with its role on muscle cell phosphate metabolism. The sterol markedly increased leucine incorporation into total cell proteins in cultured chick embryo myoblasts. This enhancement was greater than that produced by 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) and occurred prior to a significant stimulation of cell phosphate accumulation. Maximum effects of 25 OHD3 (8 h) on myoblast phosphate uptake were suppressed by cycloheximide indicating that they are mediated by de novo protein synthesis. At a similar treatment period, labelling of myoblasts with [3H]leucine (control) and [14C]leucine (+25 OHD3) followed by co-electrophoresis of total protein extracts on SDS-PAGE and isoelectrofocusing gels revealed that the sterol selectively affects the synthesis of proteins of 20 kDa and 50 kDa. These macromolecules were recovered in the microsomal fraction after differential centrifugation of homogenates. Further fractionation of myoblast microsomes on sucrose density gradients showed co-localization of the 50 kDa and 20 kDa proteins with microsomal subfractions which preferentially bind [3H-alpha]bungarotoxin, suggesting that the proteins induced by 25 OHD3 are associated to plasma membranes and may play a role in the effects of the sterol on cell phosphate uptake.

Animals↗