RESUMEN
We have previously obtained compelling proof-of-principle evidence for COX2 gene therapy for fracture repair using integrating retroviral vectors. For this therapy to be suitable for patient uses, a suitable vector with high safety profile must be used. Accordingly, this study sought to evaluate the feasibility of AAV as the vector for this COX2 gene therapy, because AAV raises less safety issues than the retroviral vectors used previously. However, an appropriate AAV serotype is required to provide early increase in and adequate level of COX2 expression that is needed for fracture repair. Herein, we reported that AAV-DJ, an artificial AAV pseudoserotype, is highly effective in delivering COX2 gene to fracture sites in a mouse femoral fracture model. Compared with AAV-2, the use of AAV-DJ led to ~5-fold increase in infectivity in mesenchymal stem cells (MSCs) and provided an earlier and significantly higher level of transgene expression at the fracture site. Injection of this vector at a dose of 7.5 × 10(11) genomic copies led to high COX2 level at the fracture site on day 3 after injections and significantly promoted fracture union at 21 days, as analyzed by radiography and µ-CT. The therapeutic effect appears to involve enhanced osteoblastic differentiation of MSCs and remodeling of callus tissues to laminar bone. This interpretation is supported by the enhanced expression of several key genes participating in the fracture repair process. In conclusion, AAV-DJ is a promising serotype for the AAV-based COX2 gene therapy of fracture repair in humans.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Dependovirus/metabolismo , Curación de Fractura , Tibia/lesiones , Transgenes , Animales , Modelos Animales de Enfermedad , Terapia Genética , Vectores Genéticos/administración & dosificación , Masculino , Ratones Endogámicos C57BLRESUMEN
Parameters of bone formation and resorption were measured in rats orbited for 19.5 days aboard the Soviet Cosmos 782 biological satellite. The most striking effects were on bone formation. During flight, rats formed significantly less periosteal bone than did control rats on the ground. An arrest line at both the periosteum and the endosteum of flight animals suggest that a complete cessation of bone growth occurred. During a 26-day postflight period, the defect in bone formation was corrected. No significant changes in bone resorption were observed.
Asunto(s)
Medicina Aeroespacial , Desarrollo Óseo , Vuelo Espacial , Animales , Matriz Ósea/fisiología , Resorción Ósea , Masculino , Periostio/fisiología , Ratas , Organismos Libres de Patógenos Específicos , Tetraciclina , Tibia/citología , Tibia/crecimiento & desarrollo , Tibia/fisiología , Factores de Tiempo , IngravidezRESUMEN
Fluoride is one of the most potent but least well understood stimulators of bone formation in vivo. Bone formation was shown to arise from direct effects on bone cells. Treatment with sodium fluoride increased proliferation and alkaline phosphatase activity of bone cells in vitro and increased bone formation in embryonic calvaria at concentrations that stimulate bone formation in vivo.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Desarrollo Óseo/efectos de los fármacos , Huesos/citología , Fluoruros/farmacología , Animales , Huesos/embriología , Huesos/enzimología , División Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Hormona Paratiroidea/farmacologíaRESUMEN
A new radioreceptor assay was used to quantify changes in serum concentration of 1alpha,25-dihydroxyvitamin D3 in rats with low calcium or low phosphate diets. Low availability of either ion elicits a fivefold increase in the circulating concentration of 1alpha,25-dihydroxyvitamin D3. The enhancement of 1alpha,25-dihydroxyvitamin D3 concentration in response to calcium deficiency is dependent on the presence of the parathyroid or thyroid glands (or both), suggesting that this effect is mediated by parathyroid hormone. In contrast, the response of phosphate deficiency is independent of these glands and may result from an action of low serum phosphate concentration or some factor associated with phosphate depletion on the renal synthesis of the 1alpha,25-dihydroxyvitamin D3 hormone.
Asunto(s)
Calcio de la Dieta , Dihidroxicolecalciferoles/sangre , Hidroxicolecalciferoles/sangre , Fosfatos/farmacología , Animales , Calcitonina/fisiología , Dieta , Riñón/fisiología , Masculino , Hormona Paratiroidea/fisiología , Fosfatos/deficiencia , RatasRESUMEN
Quantitative morphologic methods were used to measure the effects of feeding a low phosphorus diet to intact and thyroparathyroidectomized rats on several processes of bone mineralization and turnover. In severely hypophosphatemic animals, the matrix formation rate was decreased, the osteoid maturation rate was decreased, which indicated a delay in the onset of mineralization, the initial rate of mineralization was decreased, and the endosteal osteoclastic bone resorption rate was increased. In moderately hypophosphatemic animals, there was a substantial increase in bone resorption but no change in formation or in mineralization. The increase in endosteal bone resorption was due to an increase in the linear rate of bone resorption and particularly to an increase in the length of the endosteal resorbing surface. The magnitude of the increase in bone resorption was similar in thyroparathyroidectomized and intact rats indicating that neither parathyroid hormone nor calcitonin is involved in this change. This, together with the finding that there was a strong negative correlation (r = -0.99) between the per cent endosteal resorbing surface and the serum phosphorus, supports the view that the increased resorption was due to hypophosphatemia. This inverse relationship between endosteal resorbing surface and serum phosphorus appeared to hold for values of serum phosphorus above normal. The resorptive response to hypophosphatemia, as previously shown for the resorptive response to excess endogenous parathyroid hormone, was partially inhibited by vitamin D deficiency. Increased resorption occurred at levels of serum phosphorus where no changes were observed in bone formation, mineralization, or growth, suggesting that this resorptive response functions as a homeostatic mechanism to maintain serum and intracellular phosphorus concentrations.
Asunto(s)
Desarrollo Óseo , Resorción Ósea , Huesos/metabolismo , Fosfatos/sangre , Animales , Matriz Ósea/metabolismo , Calcitonina/fisiología , Calcio/metabolismo , Dieta , Homeostasis , Inyecciones Intraperitoneales , Métodos , Glándulas Paratiroides/cirugía , Hormona Paratiroidea/fisiología , Fósforo/sangre , Fósforo/metabolismo , Ratas , Tetraciclina/administración & dosificación , Tiroidectomía , Tibia/anatomía & histología , Tibia/patología , Factores de TiempoRESUMEN
In previous work we found that vitamin D-deficient and also calcium-deficient rats developed hypocalcemia and an impairment of bone formation and mineralization. The present study of thyroparathyroidectomized (TPTX) rats was undertaken to determine the effect of hypocalcemia without secondary hyperparathyroidism. TPTX rats fed a normal diet developed hypocalcemia and hyperphosphatemia in association with impairment of osteoblastic bone matrix formation and of mineralization of newly formed matrix. The serum calcium x phosphorus product was not decreased. The decreased formation was largely due to a reduction in matrix apposition indicating decreased synthetic activity of individual ostcoblasts. In contrast to the above results, when TPTX rats were fed a high-calcium diet to prevent hypocalcemia, no impairment of either formation or mineralization was found. From the results of these two experiments, it is reasonably certain that hypocalcemia was responsible for the inhibition of formation and mineralization. Moreover, based on the magnitude of the changes in serum calcium and bone parameters in TPTX rats, hypocalcemia could have accounted for the inhibition of formation and mineralization in calcium-deficient as well as vitamin D-deficient rats. In TPTX rats the mineralization defect was manifested by decreases in both the rate of osteoid maturation (indicating a delayed onset of mineralization) and the rate of mineralization. A strong correlation (r = 0.95, P < 0.001) was observed between these two rates suggesting a tight coupling of these two aspects of mineralization.TPTX rats also had lower bone resorption rates and higher serum phosphorus levels than sham-operated animals when the normal calcium diet was fed but not when the high-calcium diet was fed. Thus the inhibition of bone resorption in TPTX rats was at least partially prevented by correction of hyperphosphatemia. This is consistent with previous work showing an inverse relationship between serum phosphorus and bone resorption. Accordingly, the depression of bone resorption in TPTX rats was probably due to hyperphosphatemia as well as to hypoparathyroidism.
Asunto(s)
Desarrollo Óseo , Matriz Ósea/crecimiento & desarrollo , Resorción Ósea , Glándulas Paratiroides/fisiología , Glándula Tiroides/fisiología , Animales , Calcio/sangre , Calcio de la Dieta/administración & dosificación , Dieta , Hipocalcemia/fisiopatología , Masculino , Osteoclastos , Fósforo/sangre , Ratas , Tiroidectomía , Deficiencia de Vitamina D/fisiopatologíaRESUMEN
Quantitative histologic methods have been devised to measure several processes dealing with formation and mineralization of matrix and bone resorption. In vitamin D-deficient rats, the total osteoblastic matrix formation rate was 20% less and the total osteoclastic bone resorption rate was 80% more than in pair-fed control rats. These changes were found to be primarily because of changes in the rates of matrix formation and of bone resorption per unit area of forming or resorbing surfaces rather than to changes in the areas of these surfaces. The rate of maturation of osteoid and the rate of initial mineralization both were reduced to half of normal in the vitamin D-deficient rats. These variables related to matrix formation and mineralization were significantly correlated with the concentration of calcium but not with the concentration of phosphate in serum. The occurrence of hypocalcemia is interpreted as the consequence, both of reduced calcium absorption and of inadequate resorptive response of bone cells to homeostatic stimuli, such that, although bone resorption was greater than normal, it did not adequately compensate for the reduced intestinal absorption.
Asunto(s)
Desarrollo Óseo , Resorción Ósea , Huesos/fisiopatología , Deficiencia de Vitamina D/fisiopatología , Animales , Calcificación Fisiológica , Hipocalcemia/fisiopatología , Microscopía Fluorescente , Minerales/análisis , Fósforo/metabolismo , Ratas , TetraciclinaRESUMEN
A competitive protein binding assay for measurement of the plasma concentration of 1 alpha, 25-dihydroxyvitamin D3 [1alpha, 25-(OH)2D3] has been extended to include the immediate precursor of this hormone, 25-hydroxyvitamin D3 (25-OHD3). In addition, the assay system is capable of measuring the two metabolic products of ergocalciferol, namely. 25-hydroxyvitamin D2 (25-OHD2) and 1alpha, 25-dihydroxyvitamin D2 [1alpha, 25-(OH)2D2]. The target tissue assay system consists of a high affinity cytosol receptor protein that binds the vitamin D metabolites and a limited number of acceptor sites on the nuclear chromatin. By utilizing a series of chromatographic purification steps, a single plasma sample can be assayed for any of the four vitamin D metabolites either individually or combined. Therefore, the assay procedure allows for both the quantitative and qualitative assessment of the total active vitamin D level in a given plasma sample. To show that the binding assay was capable of measuring 1alpha, 25-(OH)2D2 as well as 1alpha, 25 (OH)2D3, two groups of rats were raised. One group, supplemented with vitamin D3, produced assayable material that represented 1alpha, 25-(OH)2D3. The other group, fed only vitamin D2 in the diet, yielded plasma containing only 1alpha, 25-(OH)2D2 as the hormonal form of the vitamin. The circulating concentrations of the two active sterols were nearly identical (15 ng/100 ml) in both groups, indicating that the competitive binding assay can be used to measure both hormonal forms in plasma. In a separate experiment, 1alpha, 25-(OH)2D2 was generated in an in vitro kidney homogenate system using 25-OHD2 as substrate. Comparison of this sterol with 1alpha, 25-(OH)2D3 in the assay system showed very similar binding curves; the D2 form was slightly less efficient (77%). Comparison of the respective 25-hydroxy forms (25-OHD2 vs. 25-OHD3) at concentrations 500-fold that of 1alpha, 25-(OH)2D3, again suggested that the binding of the D2 metabolite was slightly less efficient (71%). Finally, the assay was employed to measure the total active vitamin D metabolite pools in the plasma of normal subjects and patients with varying degrees of hypervitaminosis D. The normal plasma levels of 25-OHD and 1alpha, 25-(OH)2D measured in Tucson adults were 25-40 ng/ml and 2.1-4.5 ng/100 ml, respectively. Both sterols were predominately (greater than 90%) in the form of vitamin D3 metabolites in this environment. Typical cases of hypervitaminosis D exhibited approximately a 15-fold increase in the plasma 25-OHD concentration, and a dramatic changeover to virtually all metabolites existing in the form of D2 vitamins. In contrast, the circulating concentration of 1alpha, 25-(OH)2D was not substantially enhanced in vitamin D-intoxicated patients. We therefore conclude that hypervitaminosis D is not a result of abnormal plasma levels of 1alpha, 25-(OH)2D but may be cuased by an excessive circulating concentration of 25-OHD.
Asunto(s)
Dihidroxicolecalciferoles/sangre , Ergocalciferoles/análogos & derivados , Hidroxicolecalciferoles/sangre , Vitamina D/efectos adversos , Animales , Unión Competitiva , Pollos , Cromatografía , Dihidroxicolecalciferoles/metabolismo , Ergocalciferoles/sangre , Ergocalciferoles/metabolismo , Humanos , Hidroxicolecalciferoles/metabolismo , Masculino , Unión Proteica , Ensayo de Unión Radioligante , RatasRESUMEN
Osteoinduction is the formation of ectopic bone that follows implantation of demineralized allogeneic bone matrix (DABM) and is believed to be secondary to the release of associated inductive factors from bone matrix. To clarify the role of vitamin D in osteoinduction, we implanted DABM from vitamin D-deficient rats (-D rats) into normal rats (+D rats). Because mitogens and osteocalcin might be involved in osteoinduction, these were measured. Mitogenic activity in extracts from mineralized allogeneic bone matrix (ABM) and DABM from both +D and -D rats was determined with an assay that utilizes monolayer cultures of embryonic chick calvarial cells. Osteocalcin in serum and DABM was measured by radioimmunoassay. DABM from -D rats did not promote osteoinduction as effectively as DABM from +D rats. Resorption of implant matrix from -D rats was diminished compared with resorption of matrix from +D rats (P less than 0.01), and the decrease was attributed to a corresponding decrease in the number of osteoclasts in the implants (P less than 0.02). Bone formation (P less than 0.01) and total implant mineralization (P less than 0.001) were significantly reduced in implants from -D rats, and the reductions corresponded with a decline in the number of osteoblasts (P less than 0.05). Mitogenic activity in DABM from +D rats was only slightly decreased as compared with activity in ABM, but DABM from -D rats contained significantly less activity (P less than 0.001). No mitogenic activity was identified in implants of DABM from either +D or -D rats 3 wk after implantation. Serum osteocalcin was significantly higher in -D as compared with +D animals. In contrast, the concentrations of osteocalcin in DABM from the two groups of animals were not significantly different from each other. These findings indicate that the diminished osteoinductive activity of DABM from -D rats results from deficiency of one or more mitogenic factors that are essential for inducing the proliferation and differentiation of bone cells at the implant site and that osteocalcin does not play a role in this regard.
Asunto(s)
Matriz Ósea/fisiología , Minerales/metabolismo , Mitógenos/fisiología , Osteogénesis , Deficiencia de Vitamina D/metabolismo , Animales , Matriz Ósea/patología , Matriz Ósea/trasplante , Resorción Ósea , Proteínas de Unión al Calcio/sangre , Sangre Fetal/fisiología , Mitógenos/análisis , Osteocalcina , Ratas , Ratas Endogámicas , Timidina/metabolismo , Deficiencia de Vitamina D/patología , Deficiencia de Vitamina D/fisiopatologíaRESUMEN
The present study determined the effects of 1,25-dihydroxycholecalciferol on serum immunoactive parathyroid hormone and on parathyroid hormone secretion in vitro. Rats injected i.p. with 1,25-dihydroxycholecalciferol, 130 pmol (2 U)/140 g body wt, which is probably a physiologic dose, had a significant 43% decrease in serum immunoreactive parathyroid hormone at 4 h. In addition, this dose of 1,25-dihydroxycholecalciferol inhibited the serum immunoreactive parathyroid hormone response to hypocalcemia induced by phosphate injection. Because the increment in serum immunoreactive parathyroid hormone was less but the decrement in serum calcium more in phosphate plus 1,25-dihydroxycholecalciferol-treated than in phosphate plus vehicle-treated rats, the impaired serum immunoreactive parathyroid hormone response to 1,25-dihydroxycholecalciferol could not be attributed to the change in serum calcium. In studies of parathyroid hormone secretion from bovine parathyroid tissue in vitro, the concentration of 1,25-dihydroxycholecalciferol used for most experiments was 1nM, which is in the range found in rat serum. 1,25-Dihydroxycholecalciferol at 1 or 100 nM significantly inhibited parathyroid hormone secretion when medium calcium concentration was normal (1.5 mM), high (3.0 mM), and low (1.0 mM). Maximum inhibition ranged from 19 to 74%; inhibition was generally seen after 2 h of incubation; and inhibition was sustained or progressive thereafter. Vitamin A, 0.1 muM, caused a marked stimulation of parathyroid hormone secretion. 1,25-Dihydroxycholecalciferol at 1 nM markedly reduced (44%) the effect of vitamin A to stimulate parathyroid hormone secretion. This effect of 1,25-dihydroxycholecalciferol was maximal at 1 h and persisted thereafter. Another steroid, hydrocortisone, 10 muM, did not inhibit parathyroid hormone secretion, suggesting that the 1,25-dihydroxycholecalciferol effect was not a nonspecific inhibitory effect on parathyroid cells. Because other workers have shown that parathyroid hormone directly stimulates 1,25-dihydroxycholecalciferol secretion, our results are consistent with the concept that there is a feedback loop where parathyroid hormone directly stimulates secretion of 1,25-dihydroxycholecalciferol, which in turn directly inhibits secretion of parathyroid hormone.
Asunto(s)
Dihidroxicolecalciferoles/farmacología , Hidroxicolecalciferoles/farmacología , Hormona Paratiroidea/metabolismo , Animales , Calcio/sangre , Bovinos , Depresión Química , Hidrocortisona/farmacología , Técnicas In Vitro , Masculino , Hormona Paratiroidea/sangre , Hormona Paratiroidea/inmunología , Ratas , Factores de Tiempo , Vitamina D/metabolismoRESUMEN
Recent studies support the concept that IGF-binding protein-5 (IGFBP-5) stimulates bone formation, at least in part, via IGF-independent mechanisms. To evaluate this hypothesis further, we evaluated in vitro and in vivo effects of IGFBP-5 on bone formation parameters using the IGF-I knockout (KO) mouse. Treatment of serum-free cultures of osteoblast clones derived from IGF-I KO mice with recombinant human IGFBP-5 increased both proliferation and alkaline phosphatase (ALP) activity in a dose-dependent manner, an effect comparable to that seen with IGF-I. IGF-II levels from media conditioned by osteoblasts derived from IGF-I KO mouse were below those detectable by RIA. To eliminate possible actions of IGF-II, if any was produced by osteoblasts derived from IGF-I knockout mice, the IGFBP-5 effect was studied in the presence of exogenously added IGFBP-4, a potent inhibitor of IGF-II actions in bone cells. Addition of IGFBP-4 blocked IGF-I- but not IGFBP-5-induced cell proliferation in osteoblasts derived from IGF-I knockout mice. Consistent with in vitro results, a single local injection of IGFBP-5 to the outer periosteum of the parietal bone of IGF-I KO mice increased ALP activity and osteocalcin levels of calvarial bone extracts. The magnitudes of IGFBP-5-induced increases in ALP and osteocalcin in parietal bone extracts of IGF-I KO mice were comparable to those seen in C3H mice. In contrast to IGFBP-5, local administration of IGFBP-4 had no significant effect on bone formation in C3H and IGF-I KO mice. These results provide the first direct evidence to our knowledge that IGFBP-5 functions as a growth factor that stimulates its actions in part via an IGF-independent mechanism.
Asunto(s)
Sustancias de Crecimiento/fisiología , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/fisiología , Fosfatasa Alcalina/metabolismo , Animales , Secuencia de Bases , División Celular/efectos de los fármacos , Células Cultivadas , Cartilla de ADN/genética , Sustancias de Crecimiento/farmacología , Humanos , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor II del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
The findings that sex-specific effects on femoral structure and peak bone mineral density (BMD) are linked to quantitative trait loci (QTL) provide evidence for the involvement of specific genes that contribute to gender variation in skeletal phenotype. Based on previous findings that the BMD QTL in chromosome 1 (Chr 1) exerts a sex-specific effect on femoral structure, we predicted that congenic sublines of mice that carry one or more of the Chr 1 BMD loci would exhibit gender difference in the volumetric BMD (vBMD) phenotype. To test this hypothesis, we compared skeletal parameters of male and female of five C57BL/6J (B6).CAST/EiJ (CAST)-1 congenic sublines of mice that carry overlapping CAST chromosomal segments from the vBMD loci in Chr 1. Femur vBMD measurements were performed by the peripheral quantitative computed tomography in male and female mice at 16 weeks of age. The skeletal phenotype of the C175-185 and C178-185 congenic sublines of mice provided evidence for the presence of the BMD1-4 locus at 178-180 Mb from the centromere. This QTL affects femur vBMD only in female mice. In contrast, CAST chromosomal region carrying BMD1-1 locus increased femur vBMD both in male and female mice. Furthermore, a gender specific effect on BMD of femur mid-shaft region (mid-BMD) was identified at 168-176 Mb in Chr 1 (F=16.49, P=0.0002), while no significant effect was found on total femur BMD (F=2.67, P=0.11). Moreover, this study allowed us to locate a body weight QTL at 168-172 Mb of Chr 1, the effect of this locus was altered in female mice that carry CAST chromosomal segment 168-176 Mb of Chr 1. Based on this study, we conclude that Chr 1 carries at least two vBMD gender-dependent loci; one genetic locus at 178-180 Mb (BMD1-4 locus) which affects both mid-shaft and total femur vBMD in female mice only, and another gender-dependent locus at 168-176 Mb (BMD1-2 locus) which affects femur mid-shaft vBMD in female but not male mice.
Asunto(s)
Densidad Ósea/genética , Cromosomas de los Mamíferos/genética , Fémur/fisiología , Sitios de Carácter Cuantitativo/genética , Animales , Peso Corporal/genética , Femenino , Masculino , Ratones , Ratones Congénicos , Fenotipo , Factores Sexuales , Tomografía Computarizada por Rayos XRESUMEN
The roles of insulin-like growth factors (IGFs) in regulating growth and their modulation by six IGF binding proteins (IGFBP) are well established. IGFBP-5, the most abundant IGFBP stored in bone, is an important regulator of bone formation via IGF-dependent and -independent mechanisms. Two new proteins, four and a half lim (FHL)-2, a transcription modulator that interacts with IGFBP-5, and a disintegrin and metalloprotease (ADAM)-9, an IGFBP-5 protease, have been identified as potential regulators of IGFBP-5 action in bone. We tested the hypothesis that agents which modulate bone formation by regulating IGFBP-5 expression would also regulate FHL-2 and ADAM-9 expression in a coordinated manner. We evaluated the expression of IGFBP-5, FHL-2, and ADAM-9 by real-time reverse transcriptase (RT)-PCR during differentiation of mouse bone marrow stromal cells into osteoblasts and in response to treatment with bone formation modulators in the LSaOS human osteosarcoma cell line. IGFBP-5 and FHL-2 increased 4.3- and 3.0-fold (P < or = 0.01), respectively, during osteoblast differentiation. Dexamethasone (Dex), an inhibitor of bone formation, decreased IGFBP-5 and FHL-2 and increased ADAM-9 in LSaOS cells (P < or = 0.05). Bone morphogenic protein (BMP)-7, a stimulator of bone formation, increased IGFBP-5 and decreased ADAM-9 (P<0.01). To determine if BMP-7 would eliminate Dex inhibition of IGFBP-5, cells were treated with Dex+BMP-7. The BMP-7-induced increase in IGFBP-5 was reduced, but not eliminated, in the presence of Dex (P < or = 0.01), indicating that BMP-7 and Dex may regulate IGFBP-5 via different mechanisms. Transforming growth factor (TGF)-beta, a stimulator of bone formation, increased IGFBP-5 and FHL-2 expression (P < or = 0.01). IGF-I and TNF-alpha decreased expression of ADAM-9 (P<0.05). In conclusion, our findings are consistent with the hypothesis that FHL-2 and ADAM-9 are important modulators of IGFBP-5 actions and are, in part, regulated in a coordinated manner in bone.
Asunto(s)
Proteínas ADAM/metabolismo , Desintegrinas/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Osteoblastos/metabolismo , Factores de Transcripción/metabolismo , Animales , Médula Ósea/metabolismo , Proteína Morfogenética Ósea 7 , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/farmacología , Diferenciación Celular , Humanos , Proteínas con Homeodominio LIM , Ratones , Células del Estroma/metabolismo , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
The SK-Luci-6 cell line, established from a large-cell anaplastic lung tumor of a patient with humoral hypercalcemia of malignancy (HHM), was investigated to identify osteolytic factors produced that might mediate HHM. Most HHM-associated tumors are thought to produce parathyroid hormone-related proteins or transforming growth factor (TGF) alpha. SK-Luci-6 cells formed s.c. tumors and induced hypercalcemia in athymic nude mice. Serum-free conditioned medium from SK-Luci-6 cultures induced bone resorption in neonatal mouse calvariae in vitro, and also contained TGF-beta activity and mitogenic activity. SK-Luci-6 cell conditioned medium did not displace [125I]epidermal growth factor binding to cell receptors or stimulate cyclic AMP formation in rat osteosarcoma cells, suggesting that the conditioned medium did not contain TGF-alpha or parathyroid hormone-related proteins. The osteolytic, TGF-beta, and mitogenic activities copurified in several chromatographic separations: gel filtration in acid and then in guanidine HCl; ion exchange; and reverse phase. The results suggest that in the HHM-associated SK-Luci-6 tumor, the causative osteolytic factor produced by the tumor cells is not a parathyroid hormone-related protein or TGF-alpha but, rather, may be a TGF-beta.
Asunto(s)
Hipercalcemia/metabolismo , Neoplasias Pulmonares/análisis , Factores de Crecimiento Transformadores/aislamiento & purificación , Animales , Calcio/análisis , Línea Celular , Embrión de Pollo , AMP Cíclico/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Desnudos , Osteólisis , Osteosarcoma/metabolismoRESUMEN
Prostatic cancer typically produces osteoblastic metastases which are not attended by marrow fibrosis (i.e., osteoblast but not stromal fibroblast proliferation). In the present study we sought to test the hypothesis that prostatic cancer cells produce factor(s) which act selectively on human osteoblasts. Such a paracrine mechanism would explain the observed increase in osteoblasts, unaccompanied by an increase in marrow fibroblasts. To test this hypothesis we investigated the mitogenic activity released by the human prostatic tumor cell line, PC3. PC3 cells have been reported previously to produce mitogenic activity for cells that was relatively specific for rat osteoblasts compared to rat fibroblasts. However, the effects of this activity on human cells has not been examined previously. PC3-conditioned medium (CM) (5-50 micrograms CM protein/ml) stimulated human osteoblast proliferation by 200-950% yet did not stimulate human fibroblast proliferation [( 3H]thymidine incorporation). PC3 CM also increased cell numbers in human osteoblast but not fibroblast cell cultures. To determine whether the osteoblast-specific mitogenic activity could be attributed to known bone growth factors, specific assays for these growth factors were performed. PC3 CM contained 10 pg insulin-like growth factor (IGF) I, less than 2 pg IGF II, 54 pg basic fibroblast growth factor, and 16 pg transforming growth factor beta/microgram CM protein. None of these growth factors alone or in combination could account for the observed osteoblast-specific PC3 cell-derived mitogenic activity. Furthermore, when 5 micrograms/ml PC3 CM was tested in combination with maximally effective concentrations of either basic fibroblast growth factor, IGF I, IGF II, or transforming growth factor beta, it produced an additive effect suggesting that PC3 CM stimulates osteoblast proliferation by a mechanism independent of these bone mitogens. Biochemical characterization supported the hypothesis that the PC3 cell growth factor was unique from other growth factors. The PC3 growth factor did not bind to heparin and was resistant to acid as well as the reducing agent, dithiothreitol. Sephadex G-75 and fast protein liquid chromatography Mono S cation-exchange chromatography revealed the PC3-derived mitogen to be an Mr 26,000-30,000 basic protein. Therefore, we conclude that PC3 cells release a mitogen which exhibits higher specificity for human osteoblasts than human fibroblasts and is unique from other growth factors tested. Production of this mitogen by human prostatic carcinoma cells could play an etiological role in the intense osteoblast-specific stimulation that occurs at sites of bone metastases.
Asunto(s)
Huesos/efectos de los fármacos , Mitógenos/farmacología , Osteoblastos/efectos de los fármacos , Neoplasias de la Próstata/patología , Animales , Huesos/citología , Huesos/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Cromatografía en Gel/métodos , Medios de Cultivo , ADN/biosíntesis , Dextranos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Sustancias de Crecimiento/aislamiento & purificación , Sustancias de Crecimiento/metabolismo , Sustancias de Crecimiento/farmacología , Humanos , Masculino , Ratones , Mitógenos/aislamiento & purificación , Mitógenos/metabolismo , Osteoblastos/metabolismo , Neoplasias de la Próstata/metabolismo , Estimulación Química , Timidina/metabolismo , Tritio , Células Tumorales CultivadasRESUMEN
Skeletal growth factor (SGF) activity was extracted from human bone matrix by demineralization and purified under dissociative conditions using hydroxyapatite, HPLC gel-filtration and HPLC reverse-phase chromatography. Human SGF thus purified was characterized chemically and biologically. Purified human SGF stimulated chick embryo bone cell proliferation at picomolar concentrations (half maximum at 2-3 ng/ml) and had little or no activity on other cell types tested (mouse 3T3 and normal rat kidney fibroblasts, embryonic chick intestinal and human placental cells). Human SGF did not displace 125I-labeled epidermal growth factor binding to normal rat kidney cells and did not stimulate normal rat kidney cell colony formation in soft agar. Human SGF activity was sensitive to trypsin, chymotrypsin, papain, dithiothreitol and performic acid but was resistant to heat (upto 70 degrees C), pH (3-10), cyanogen bromide, alkaline phosphatase and neuraminidase and did not bind jack bean concanavalin A or kidney bean lectin. From our chemical and biological studies it appears that human SGF is different from other known polypeptide growth factors: epidermal growth factor, fibroblast growth factor, insulin, insulin-like growth factor-I, platelet-derived growth factor and transforming growth factor.
Asunto(s)
Proteínas/análisis , Animales , Fenómenos Químicos , Química Física , Embrión de Pollo , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Factores de Crecimiento de Fibroblastos/análisis , Sustancias de Crecimiento/análisis , Sustancias de Crecimiento/farmacología , Humanos , Factor I del Crecimiento Similar a la Insulina/análisis , Factor II del Crecimiento Similar a la Insulina , Ratones , Mitógenos , Especificidad de Órganos , Péptidos/farmacología , Factor de Crecimiento Derivado de Plaquetas/análisis , Proteínas/farmacología , Ratas , Especificidad de la Especie , Factores de Crecimiento TransformadoresRESUMEN
We prepared aqueous extracts of whole femorae and tibiae of embryonic chicks. An amount of extract containing 25 microgram of protein resulted in a 500% increase in DNA synthesis in calvarial cell culture, and significant effects were detected with 5 microgram (55%). The time course for stimulation of DNA synthesis showed a peak occurring 16-20 h after addition of the extract. This matrix factor is nondialyzable, and fractionation on a column of Sephadex G-100 indicated a molecular weight of 60-80,000. At the maximum dose used, [3H]proline incorporation into total protein of calvarial cells was increased by 55%, and thus far, all fractions active in promoting DNA synthesis have been found to increase collagen synthesis in cultured chick tibiae. These data are consistent with an effect on osteoblasts as well as bone precursor cells. Extracts prepared from tibiae of 2-day-old chicks, from which the marrow had been removed, also stimulated DNA synthesis (280% increase), thus ruling out the possibility that the factor is a relatively nonspecific nitrogen from the hematopoietic cell line. We conclude that bone matrix contains a substance which could regulate bone formation in vitro by control of mitosis in osteogenic precursors and/or stimulation of osteoblast activity.
Asunto(s)
Huesos/fisiología , Extractos de Tejidos/farmacología , Animales , Médula Ósea/fisiología , División Celular/efectos de los fármacos , Embrión de Pollo , Replicación del ADN/efectos de los fármacos , Cinética , Técnicas de Cultivo de Órganos , Prolina/metabolismo , Biosíntesis de ProteínasRESUMEN
Human skeletal growth factor (human SGF) extracted from human bone has been purified to homogeneity by hydroxyapatite chromatography and gel filtration under dissociative conditions followed by FPLC heparin-Sepharose affinity chromatography and reverse phase HPLC. Human SGF was homogeneous except that in each preparation about 30% of SGF molecules lacked the N-terminal alanine. 75% of the human SGF sequence has been determined. The amino acid sequences of the N-terminal 20 amino acids and of several tryptic fragments were identical to the corresponding sequences of human insulin-like growth factor-II (IGF-II) purified from serum. However, since the C-peptide (variable region) of human SGF has not yet been sequenced, we cannot conclude that SGF is identical to IGF-II. Comparison of the amino acid sequence of human SGF with that of IGF-II variants that have been described in the literature revealed that human SGF is not one of the known IGF-II variants. IGF-I was also found in human bone extract but was several-fold less abundant than SGF/IGF-II. The relative abundance of SGF/IGF-II and IGF-I in bone corresponded to the relative rates of production of these two mitogens by human bone cells in vitro. Regarding the physiological significance of IGF-II in bone, previous studies on the biological actions of SGF in vitro suggest that this growth factor can have both paracrine and autocrine functions on cells of the osteoblast line. In addition, we have proposed the concept that SGF is a mediator of the coupling of bone formation to bone resorption, an important bone volume regulatory mechanism. In as much as SGF is very similar (if not identical) to IGF-II, it seems likely that these proposed regulatory functions of SGF in bone are attributable to IGF-II.
Asunto(s)
Sustancias de Crecimiento/aislamiento & purificación , Factor II del Crecimiento Similar a la Insulina/análisis , Proteínas/aislamiento & purificación , Somatomedinas/análisis , Secuencia de Aminoácidos , Huesos/efectos de los fármacos , Huesos/metabolismo , Cromatografía de Afinidad , Cabeza Femoral/análisis , Heparina/metabolismo , Humanos , Datos de Secuencia Molecular , Peso Molecular , Proteínas/metabolismo , Proteínas/farmacología , Homología de Secuencia de Ácido Nucleico , Timidina/metabolismoRESUMEN
Human bone cells isolated from femoral heads were cultured in BGJb medium containing bovine serum albumin (100 micrograms/ml), insulin (1 microgram/ml) and epidermal growth factor (10 ng/ml), and the conditioned medium collected. The medium was concentrated, chromatographed using HPLC gel filtration (TSK 2000 SW), and assayed for mitogenic activity using [3H]thymidine incorporation into embryonic chick calvarial cells. The conditioned medium contained mitogenic activity which eluted with a different elution time than insulin or epidermal growth factor. Characterization of this activity suggests that it was due to human skeletal growth factor (SGF), a mitogen which had been previously isolated from human bone matrix. Common properties include: stimulation of DNA synthesis in cultured embryonic chick calvarial cells, competition with human SGF for binding to anti-SGF antibodies, elution from HPLC gel filtration as a large factor (Mr 100,000) under native conditions but as a small factor (Mr 10,000) under dissociative conditions (4 M guanidine HCl), elution time on HPLC reverse-phase chromatography (small SGF), inactivation by dithiothreitol, stability to heat, acidic or alkaline conditions and inactivation by trypsin and chymotrypsin. These observations provide evidence that human bone cells produce SGF. Conditioned medium from human skin cell cultures also contained mitogenic activity. However, the activity was less than that from bone cells and did not cross-react with the rat anti-SGF antibodies.
Asunto(s)
Osteoblastos/metabolismo , Biosíntesis de Proteínas , Proteínas , Animales , Unión Competitiva , Bioensayo , Células Cultivadas , Embrión de Pollo , Cromatografía Líquida de Alta Presión , Quimotripsina/metabolismo , Replicación del ADN/efectos de los fármacos , Ditiotreitol/farmacología , Ensayo de Inmunoadsorción Enzimática , Humanos , Concentración de Iones de Hidrógeno , Factor II del Crecimiento Similar a la Insulina , Peso Molecular , Osteoblastos/citología , Temperatura , Tripsina/metabolismoRESUMEN
In order to investigate the mechanism(s) of electric field-stimulated osteogenesis, we have developed an in vitro model in which embryonic chick tibiae have consistently demonstrated increased bone matrix formation in response to a low amplitude (estimated 10(-5) V/m in the serum-free culture medium), capacitively coupled, 10 Hz sinusoidal electric field. Initial applications of this model revealed that 72 h of continuous exposure to the electric field increased tibial collagen production by 29% compared to untreated controls, P less than 0.01. Additional studies further revealed: (a) that when electric field exposure was limited to 30 min/day during the 72 h in vitro incubation, embryonic bone matrix formation was increased by 83%, compared to non-treated controls (P less than 0.001), suggesting an inductive mechanism; (b) that the osteogenic response to electric field exposure in vitro was not unique to embryonic chick tibiae, since a similar response was also seen with newborn mouse calvaria (+133%, P less than 0.02); (c) that electric field-exposure-stimulated chick bone matrix formation was associated with increased bone cell proliferation; and (d) that this mitogenic response to in vitro electric field exposure could also be observed with embryonic chick calvarial cells in monolayer, serum-free cultures.