RESUMEN

BACKGROUND AND OBJECTIVES: Lycopene is a carotenoid commonly found in tomatoes and tomato products which acts as an antioxidant to decrease oxidative stress and osteoporosis risk. We wanted to determine the effects of a lycopene-restricted diet on oxidative stress parameters and bone turnover markers in postmenopausal women. SETTING: St. Michael 's Hospital, Toronto, ON, Canada. PARTICIPANTS AND STUDY DESIGN: 23 healthy postmenopausal women, 50-60 years old, provided blood samples at baseline and following a one-month lycopene-depletion period. MEASUREMENTS: Serum samples were analyzed for carotenoids; the oxidative stress parameters protein thiols and thiobarbituric-malondialdehyde reactive substances; the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and the bone turnover markers bone alkaline phosphatase and crosslinked N-telopeptide of type I collagen (NTx). A paired t-test was used to test for significant differences in bone turnover markers, oxidative stress parameters and antioxidant status after lycopene restriction. RESULTS: Dietary lycopene restriction resulted in significantly decreased serum lycopene (p < 0.0001), lutein/zeaxanthin (p < 0.01), and α -/ß -carotene (p < 0.05). GPx (p < 0.01), lipid and protein oxidation increased (not significant), while CAT and SOD were significantly depressed (p < 0.05 and p < 0.005, respectively). These changes coincided with significantly increased NTx (p < 0.05). CONCLUSIONS: These findings suggest that the daily consumption of lycopene may be important as it acts as an antioxidant to decrease bone resorption in postmenopausal women and may therefore be beneficial in reducing the risk of osteoporosis.

Asunto(s)

Resorción Ósea/sangre , Carotenoides/administración & dosificación , Carotenoides/sangre , Dieta , Osteoporosis Posmenopáusica/sangre , Estrés Oxidativo , Antioxidantes/administración & dosificación , Antioxidantes/metabolismo , Biomarcadores/sangre , Resorción Ósea/epidemiología , Estudios Cruzados , Femenino , Glutatión Peroxidasa/metabolismo , Humanos , Peroxidación de Lípido , Licopeno , Malondialdehído/metabolismo , Persona de Mediana Edad , Osteoporosis Posmenopáusica/prevención & control , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Superóxido Dismutasa/metabolismo

RESUMEN

UNLABELLED: To date, no intervention studies have been published demonstrating the effect of the antioxidant lycopene on bone. Postmenopausal women supplemented with lycopene had significantly increased antioxidant capacity and decreased oxidative stress and the bone resorption marker N-telopeptide (NTx). Lycopene decreases bone resorption markers and may reduce the risk of osteoporosis. INTRODUCTION: We have previously shown in vitro and in vivo that lycopene from tomato is associated with a protective effect on bone, but lycopene intervention studies have not been reported. Our aim was to carry out a randomized controlled intervention study to determine whether lycopene would act as an antioxidant to decrease oxidative stress parameters, resulting in decreased bone turnover markers, thus reducing the risk of osteoporosis in postmenopausal women. METHODS: Sixty postmenopausal women, 50-60 years old, were recruited. Following a 1-month washout without lycopene consumption, participants consumed either (N = 15/group): (1) regular tomato juice, (2) lycopene-rich tomato juice, (3) tomato Lyc-O-Mato lycopene capsules, or (4) placebo capsules, twice daily for total lycopene intakes of 30, 70, 30, and 0 mg/day respectively for 4 months. Serum collected after the washout, 2 and 4 months of supplementation, was assayed for cross-linked aminoterminal N-telopeptide, carotenoid content, total antioxidant capacity (TAC), lipid, and protein oxidation. RESULTS: Participants who consumed juice or lycopene capsules were analyzed in one group designated "LYCOPENE-supplemented". Repeated measures ANOVA showed that LYCOPENE-supplementation for 4 months significantly increased serum lycopene compared to placebo (p < 0.001). LYCOPENE-supplementation for 4 months resulted in significantly increased TAC (p < 0.05) and decreased lipid peroxidation (p < 0.001), protein oxidation (p < 0.001), and NTx (p < 0.001). These decreases in lipid peroxidation, protein oxidation, and NTx were significantly different from the corresponding changes resulting from placebo supplementation (p < 0.05, p < 0.005, and p < 0.02, respectively). CONCLUSIONS: Our findings suggest that the antioxidant lycopene is beneficial in reducing oxidative stress parameters and the bone resorption marker NTx.

Asunto(s)

Antioxidantes/uso terapéutico , Resorción Ósea/tratamiento farmacológico , Carotenoides/uso terapéutico , Suplementos Dietéticos , Estrés Oxidativo/efectos de los fármacos , Antioxidantes/metabolismo , Bebidas , Biomarcadores/sangre , Resorción Ósea/sangre , Carotenoides/análisis , Carotenoides/sangre , Colágeno Tipo I/sangre , Femenino , Humanos , Licopeno , Solanum lycopersicum , Persona de Mediana Edad , Osteoporosis Posmenopáusica/prevención & control , Péptidos/sangre , Posmenopausia/sangre , Posmenopausia/fisiología

RESUMEN

BACKGROUND/AIMS: Polymorphisms of the paraoxonase 1 (PON1) enzyme affect the ability to protect LDL from oxidation. Oxidative stress is a risk factor for osteoporosis and antioxidants may be beneficial for prevention. The aim of this study was to determine whether PON1 genotypes modified the association between lycopene and bone turnover markers and oxidative stress parameters. METHODS: Blood samples from 107 women 25-70 years of age were analyzed for serum carotenoid concentrations, bone-specific alkaline phosphatase (BAP), N-telopeptide of type I collagen (NTx) and oxidative stress parameters. Subjects were genotyped for the 172T→A and 584A→G polymorphisms of PON1. RESULTS: The 172T→A polymorphism modified the association between lycopene and NTx (p < 0.05 for interaction). In the 172TT genotype, high serum lycopene was associated with decreased NTx (p < 0.05). The 584A→G polymorphism modified the association between lycopene and BAP (p < 0.05 for interaction). Additionally, in participants with the 584GG genotype, high serum lycopene was associated with high TBA-reactive substances (p < 0.05). CONCLUSIONS: These findings show that PON1 polymorphisms modify the association between serum concentrations of lycopene and oxidative stress parameters and bone turnover markers and may, therefore, moderate the risk of osteoporosis.

Asunto(s)

Arildialquilfosfatasa/genética , Huesos/metabolismo , Carotenoides/sangre , Estrés Oxidativo/fisiología , Polimorfismo Genético , Polimorfismo de Nucleótido Simple , Adulto , Anciano , Fosfatasa Alcalina/sangre , Colágeno Tipo I/sangre , Registros de Dieta , Femenino , Humanos , Licopeno , Persona de Mediana Edad , Péptidos/sangre

RESUMEN

Lycopene is an antioxidant associated with a reduced risk of chronic diseases common in women such as osteoporosis and cancer; however, no official recommendation for lycopene consumption exists, and intake data from Canadian women are limited. This study was designed to generate information about average lycopene intake in Canadian women of different ages. A cross-sectional study was conducted at St. Michael's Hospital in Toronto, Ontario, Canada. One hundred one women, between the ages of 25 and 70 years, who were not on any medications, were recruited to record their diet for 7 days. Statistical analyses were performed to compare the types of lycopene-containing foods consumed, associations between intake of lycopene and macro/micronutrients, and whether participant characteristics, such as body mass index, could predict lycopene intake. Average lycopene intake was 6.14 +/- 5.35 mg/day, which is higher than reported in other countries. Intake was similar among age groups but was highly variable. Raw tomatoes were the most frequently consumed source of lycopene, while participants with the highest lycopene intake consumed more cooked/processed tomato products than those with lower intake (P < .005). Participants 25-49 years old consumed more dried/powdered tomatoes (P < .05), pizza (P < .002), and ketchup (P < .10) than 50-70 year olds. Lycopene intake could not be predicted by any participant characteristics. In older participants, lycopene intake was positively correlated with intake of calcium, niacin, and vitamins A, D, and K (P < or = .05). These findings are significant to women's health and may contribute to the establishment of nutritional and health recommendations regarding consumption of lycopene by Canadian women to prevent chronic diseases.

Asunto(s)

Antioxidantes/administración & dosificación , Carotenoides/administración & dosificación , Dieta , Adulto , Factores de Edad , Anciano , Calcio de la Dieta/administración & dosificación , Canadá , Estudios Transversales , Dieta/etnología , Femenino , Humanos , Licopeno , Solanum lycopersicum , Persona de Mediana Edad , Vitaminas/administración & dosificación

RESUMEN

Oxidative stress is an important contributor to the risk of chronic diseases. Dietary guidelines recommend increased consumption of fruits and vegetables to combat the incidence of human diseases such as cancer, cardiovascular disease, osteoporosis and diabetes. Fruits and vegetables are good sources of antioxidant phytochemicals that mitigate the damaging effect of oxidative stress. Carotenoids are a group of phytochemicals that are responsible for different colors of the foods. They are recognized as playing an important role in the prevention of human diseases and maintaining good health. In addition to being potent antioxidants some carotenoids also contribute to dietary vitamin A. There is scientific evidence in support of the beneficial role of phytochemicals in the prevention of several chronic diseases. Although the chemistry of carotenoids has been studied extensively, their bioavailability, metabolism and biological functions are only now beginning to be investigated. Recent interest in carotenoids has focused on the role of lycopene in human health. Unlike some other carotenoids, lycopene does not have pro-vitamin A properties. Because of the unsaturated nature of lycopene it is considered to be a potent antioxidant and a singlet oxygen quencher. This article will review carotenoids in general and lycopene in particular for their role in human health.

Asunto(s)

Carotenoides/farmacología , Animales , Antioxidantes/farmacología , Disponibilidad Biológica , Enfermedades Cardiovasculares/prevención & control , Carotenoides/administración & dosificación , Carotenoides/química , Humanos , Licopeno , Neoplasias/prevención & control , Osteoporosis/prevención & control

RESUMEN

INTRODUCTION: Oxidative stress induced by reactive oxygen species (ROS) is associated with the risk of osteoporosis, and can be reduced by certain dietary antioxidants. Lycopene is an antioxidant known to decrease the risk of age-related chronic diseases, such as cancer. However, the role of lycopene in osteoporosis has not yet been investigated. MATERIALS AND METHODS: In a cross-sectional study, 33 postmenopausal women aged 50-60 years provided seven-day dietary records and blood samples. Serum samples were used to measure serum lycopene, lipid peroxidation, protein thiols, bone alkaline phosphatase (BAP), and cross-linked N-telopeptides of type I collagen (NTx). The serum lycopene per kilogram body weight of the participants was grouped into quartiles and associated with the above serum parameters using one-way ANOVA and the Newman-Keuls post-test. RESULTS: The results showed that groups with higher lycopene intake, as determined from the dietary records, had higher serum lycopene (p<0.02). A higher serum lycopene was found to be associated with a low NTx (p<0.005). Similarly, groups with higher serum lycopene had lower protein oxidation (p<0.05). DISCUSSION: In conclusion, these results suggest that the dietary antioxidant lycopene reduces oxidative stress and the levels of bone turnover markers in postmenopausal women, and may be beneficial in reducing the risk of osteoporosis.

Asunto(s)

Antioxidantes/administración & dosificación , Resorción Ósea/prevención & control , Carotenoides/administración & dosificación , Estrés Oxidativo/efectos de los fármacos , Antioxidantes/metabolismo , Composición Corporal/fisiología , Peso Corporal/fisiología , Carotenoides/sangre , Cromatografía Líquida de Alta Presión/métodos , Estudios Transversales , Registros de Dieta , Femenino , Humanos , Peroxidación de Lípido/efectos de los fármacos , Licopeno , Persona de Mediana Edad , Osteoporosis Posmenopáusica/prevención & control

RESUMEN

Oxidative stress is now recognized as an important etiological factor in the causation of several chronic diseases including cancer, cardiovascular diseases, osteoporosis, and diabetes. Antioxidants play an important role in mitigating the damaging effects of oxidative stress on cells. Lycopene, a carotenoid antioxidant, has received considerable scientific interest in recent years. Epidemiological, tissue culture, and animal studies provide convincing evidence supporting the role of lycopene in the prevention of chronic diseases. Human intervention studies are now being conducted to validate epidemiological observations and to understand the mechanisms of action of lycopene in disease prevention. To obtain a better understanding of the role of lycopene in human health, this chapter reviews the most recent information pertaining to its chemistry, bioavailability, metabolism, role in the prevention of prostate cancer and cancer of other target organs, its role in cardiovascular diseases, osteoporosis, hypertension, and male infertility. A discussion of the most relevant molecular markers of cancer is also included as a guide to future researchers in this area. The chapter concludes by reviewing global intake levels of lycopene, suggested levels of intake, and future research directions.

Asunto(s)

Carotenoides , Animales , Antioxidantes , Disponibilidad Biológica , Enfermedades Cardiovasculares/prevención & control , Carotenoides/administración & dosificación , Carotenoides/análisis , Carotenoides/farmacocinética , Enfermedad Crónica , Dieta , Estabilidad de Medicamentos , Análisis de los Alimentos , Humanos , Hipertensión/prevención & control , Infertilidad Masculina/prevención & control , Licopeno , Masculino , Neoplasias/prevención & control , Enfermedades Neurodegenerativas/prevención & control , Osteoporosis/prevención & control , Estrés Oxidativo , Distribución Tisular

RESUMEN

We have shown that osteoblastic cells derived from trabecular bone explants of osteoporotic subjects (OP cells) exhibited an altered alkaline phosphatase (ALP) response to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] compared to control (CON) cells. Our hypothesis that OP cells have other intrinsic abnormalities was investigated using our cell models representing two different stages of differentiation. OP and CON cells were cultured in the absence (-DEX) or presence (+DEX) of 10 nM dexamethasone (DEX) in 10% fetal calf serum (FCS) prior to exposure to serum-free medium containing 1 nM of PTH and/or 17-beta estradiol (E2). Both OP and CON cells responded to DEX with a two-fold increase in basal ALP activity. While E2 or PTH+E2 had no effect on OP cells, both treatments inhibited ALP activity in CON cells (p<0.05). OP and CON cells grown in DEX also expressed PTH-stimulated adenylate cyclase (AC) activities higher than those of (-DEX) cells. OP+DEX cells, however, exhibited activities which were 8-fold higher than those of CON+DEX cells (p<0.001). In OP+DEX cells, E2 stimulated basal AC activity (p<0.05) but did not affect PTH-stimulated activity. In contrast, in CON+DEX cells, E2 had no effect on basal activity but inhibited PTH-stimulated AC activity (p<0.001). Osteocalcin production was 4-fold lower in OP+DEX cells compared to OP-DEX and CON cells (p<0.05) while osteocalcin mRNA levels were significantly lower in OP+DEX and CON+/-DEX cells compared to OP-DEX cells (p<0.05). E2 did not affect osteocalcin protein or mRNA levels in either OP or CON cells. No differences in mRNA levels were found for estrogen receptor-alpha (ER-a) in OP+/-DEX cells whereas these levels were significantly higher in CON+DEX compared to CON-DEX cells (p<0.05). These results indicate that DEX amplified the differences between OP and CON cells and confirm the presence of intrinsic osteoblastic abnormalities in patients with osteoporosis that persist in culture.

Asunto(s)

Dexametasona/farmacología , Osteoblastos/efectos de los fármacos , Osteoblastos/patología , Osteoporosis/patología , Adenilil Ciclasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Fosfatasa Alcalina/metabolismo , Calcitriol/farmacología , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteoblastos/enzimología , Osteocalcina/genética , Osteocalcina/metabolismo , Osteoporosis/enzimología , Osteoporosis/genética , Hormona Paratiroidea/farmacología , Fenotipo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/genética , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Factores de Tiempo

RESUMEN

It is now well established that estrogen inhibits bone resorption. However, its effect on bone formation remains controversial. We studied the effect of 17beta-estradiol (E2) on mineralized bone nodule formation in long-term cultures of osteosarcoma SaOS-2 cells. We showed that SaOS-2 cells formed mineralized nodules which under electron microscopy revealed a bone structure with active osteoblasts, entrapped osteocytes, extracellular collagen fibrils and hydroxyapatite deposits, making this system a valid model to study bone formation in vitro. Intermittent addition of E2 for 6 hours during a 48-hour cycle of changes of medium, starting from day 3, resulted in a dose-dependent stimulation of mineralized bone nodule number and area, as well as alkaline phosphatase activity. In conclusion, we report for the first time a stimulatory effect of E2 on mineralized bone nodule formation in human osteoblasts in culture.

Asunto(s)

Calcificación Fisiológica/efectos de los fármacos , Estradiol/farmacología , Fosfatasa Alcalina/efectos de los fármacos , Análisis de Varianza , Neoplasias Óseas/patología , Relación Dosis-Respuesta a Droga , Humanos , Microscopía Electrónica , Osteoblastos/efectos de los fármacos , Osteosarcoma/patología , Coloración y Etiquetado , Células Tumorales Cultivadas/efectos de los fármacos

RESUMEN

We compared the effects of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and its analog, 1alpha,25-dihydroxy-16-ene-vitamin D3 [1alpha,25(OH)2-16-ene-D3], as well as their interactions with 17-beta estradiol (E2) on osteoblastic function in our human normal (HOB) and osteosarcoma SaOS-2 cell models representing two different stages of differentiation, the more differentiated HOB+DEX cells and SaOS+DEX cells, and the corresponding less differentiated HOB-DEX and SaOS-DEX cells. The differential effects of 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 and the modulation by E2 on ALP activity in HOB-DEX and HOB+DEX cells were small but significant. The most significant effects were seen in SaOS+DEX cells, in which 1alpha,25(OH)2-16-ene-D3 was 100-fold more potent than 1alpha,25(OH)2D3, the maximal enhancement being exerted at 0.1 nM and 10 nM, respectively. E2 enhanced the stimulatory effects of both compounds, with ALP being increased 2-fold at 0.1 nM (p<0.001). Osteocalcin (OC) production in HOB-DEX cells was stimulated 1.3 to 1.4-fold by 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 at a concentration of 0.01 nM, with E2 inhibiting the effect of 1alpha,25(OH)2-16-ene-D3. In SaOS-DEX and SaOS+DEX cells, 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 stimulated OC production 1.6-fold at 0.1 nM with E2 slightly enhancing the effect of 1alpha,25(OH)2D3. Western blot analysis of 1alpha,25(OH)2D3 receptor (VDR) levels showed that in SaOS+DEX cells, the effect of 1alpha,25(OH)2D3 was larger than that of 1alpha,25(OH)2-16-ene-D3. These results show that 1alpha,25(OH)2-16-ene-D3 is biologically active in human osteoblasts.

Asunto(s)

Neoplasias Óseas/patología , Calcitriol/farmacología , Estradiol/farmacología , Osteoblastos/efectos de los fármacos , Osteosarcoma/patología , Adulto , Fosfatasa Alcalina/metabolismo , Western Blotting , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Masculino , Osteocalcina/biosíntesis , Células Tumorales Cultivadas

RESUMEN

We compared the separate effects of 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and its analog, 1alpha,25-dihydroxy-16ene,23yne-vitamin D3 (1alpha25(OH)2-16ene,23yne-D3), as well as their interactions with 17-beta estradiol (E2) in our human osteosarcoma SaOS-2 cell models representing two stages of differentiation, the SaOS+DEX and SaOS-DEX cells. SaOS+DEX cells have been previously shown to express higher PTH-stimulated adenylate cyclase (PTH-AC) and basal alkaline phosphatase (ALP) activities compared with SaOS-DEX cells. ALP: In SaOS+DEX cells, 0.1 nmol/L analog, but not 1alpha,25(OH)2D3, increased ALP activity 1.7-fold (p < 0.05). Instead, 1 nmol/L 1alpha,25(OH)2D3 increased ALP 1.4-fold (p < 0.05). In these cells, E2 enhanced 1alpha,25(OH)2D3-stimulated ALP activity (ANOVA, F = 51.22, p <0.0001), while inhibiting the effect of the analog. [3H]-Thymidine uptake: In SaOS+DEX cells, 1alpha,25(OH)2D3 had biphasic effects (ANOVA, F = 13.08, p < 0.0001), which were not altered by E2. In contrast, the analog was stimulatory only with E2 (ANOVA, F = 3.59, p < 0.025). Osteocalcin (OC): 1alpha,25(OH)2D3 and its analog stimulated OC production in SaOS-DEX cells with smaller effects in SaOS+DEX cells. In SaOS-DEX cells, E2 enhanced the effect of 1alpha,25(OH)2D3, but not that of the analog. PTH-AC: In SaOS-DEX cells, 100 nmol/L analog inhibited PTH-AC activities by 50% (p < 0.01), whereas 1alpha,25(OH)2D3 had little effect. In SaOS+DEX cells, both compounds inhibited PTH-AC approximately 35%. E2 inhibited the effect of the analog in SaOS-DEX cells, but enhanced the effects of both compounds in SaOS+DEX cells. These results show that the analog 1alpha,25(OH)2-16ene,23yne-D3 was effective in regulating osteoblastic function; its effects were modulated by E2 and dependent upon the stage of osteoblast differentiation.

Asunto(s)

Calcitriol/análogos & derivados , Dihidroxicolecalciferoles/farmacología , Estradiol/farmacología , Osteoblastos/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Fosfatasa Alcalina/metabolismo , Calcitriol/farmacología , Diferenciación Celular , Humanos , Osteoblastos/metabolismo , Osteocalcina/biosíntesis , Osteocalcina/efectos de los fármacos , Osteosarcoma , Hormona Paratiroidea/farmacología , Timidina/metabolismo , Células Tumorales Cultivadas

RESUMEN

We have previously shown that the combination of estrogen (E2) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] enhanced alkaline phosphatase (ALP) activity in human osteosarcoma SaOS-2 cells which had been grown in the presence of 10 nmol/L dexamethasone (SaOS + DEX cells). To determine whether this increase in ALP activity was associated with changes in receptor protein levels for E2 (ER) in individual SaOS + DEX cells, a monoclonal antibody to ER and a histochemical stain for ALP were used localize the expression of these proteins in fixed cells. Western and Northern blot analyses were used to determine whether E2 and 1,25(OH)2D3 affected immunoreactive ER protein and mRNA levels, respectively. Our results showed that immunohistochemical staining for ER was primarily nuclear, whereas histochemical staining for ALP was cytosolic. Treatment of cells with 1,25(OH)2D3, E2, or E2 + 1,25(OH)2D3 increased the levels of both ER and ALP activity, as visualized by enhanced cellular staining. Western analyses showed that 1,25(OH)2D3 and E2, separately and in combination, significantly increased ER protein levels. 1,25(OH)2D3 enhanced ER levels in a dose-dependent manner [analysis of variance (ANOVA), F = 3.91, p < 0.05]; this effect was augmented by E2 (ANOVA, F = 5.98, p < 0.005). In comparison, 17 alpha-E2 + 1,25(OH)2D3 and tamoxifen + 17 beta-E2 + 1,25(OH)2D3 did not increase ER levels compared with those obtained with 17 beta-E2 + 1,25(OH)2D3. ER mRNA levels were not significantly increased by E2, 1,25(OH)2D3, or E2 + 1,25(OH)2D3 together. In contrast, in a population of SaOS cells which had been in culture longer (approximately 40 passages more) than the previous cells, E2 + 1,25(OH)2D3 did not enhance ALP activity or ER levels above those obtained with 1,25(OH)2D3 alone. These results showed that in responsive SaOS cells, E2 enhanced both the stimulatory effects of 1,25(OH)2D3 on ALP activity and the activation of ER. Thus changes in ALP activity are associated with changes in ER levels in SaOS + DEX cells.

Asunto(s)

Fosfatasa Alcalina/metabolismo , Osteoblastos/metabolismo , Receptores de Estrógenos/metabolismo , Análisis de Varianza , Antiinflamatorios/farmacología , Anticuerpos Monoclonales , Antineoplásicos Hormonales/farmacología , Northern Blotting , Western Blotting , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Calcitriol/farmacología , División Celular/efectos de los fármacos , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Estradiol/farmacología , Humanos , Inmunohistoquímica , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Osteosarcoma/metabolismo , Osteosarcoma/patología , ARN Mensajero/metabolismo , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/genética , Células Tumorales Cultivadas

RESUMEN

We previously developed two models of human osteoblasts with distinct differentiation stages using cells derived from iliac crest trabecular bone explants cultured long term in the presence (HOB + DEX) and absence (HOB - DEX) of 10 nM dexamethasone (DEX) (Wong et al., J Bone Miner Res 1990;5:803). Using these models from 36 subjects aged 41-80 years, we examined the effects of 17 beta-estradiol (E2) on cell proliferation, osteocalcin (OC) production, alkaline phosphatase (ALP) and basal and parathyroid hormone (PTH)-stimulated adenylate cyclase activities, as well as the steady-state mRNA levels of ALP, collagen type I(COLL), OC, and receptors for E2 (ER) and PTH (PTHr). E2 alone had no effect on [3H]thymidine uptake in (HOB - DEX) cells but appeared to stimulate the uptake in (HOB + DEX) cells in a dose-dependent manner, with maximum effect at 10(-10)M (p < 0.05). However, in the presence of 10(-6)M PTH, E2 inhibited the uptake in (HOB - DEX) cells (ANOVA, KW = 18.95, p < 0.005) but stimulated the uptake in (HOB + DEX) cells (KW = 13.52, p < 0.025). E2 decreased the amount of osteocalcin in culture media from both (HOB - DEX) and (HOB + DEX) cells (p < 0.05). PTH alone or E2, alone or in combination with 10(-9)M PTH, had no effect on ALP activity in (HOB - DEX) cells. In contrast, in (HOB + DEX) cells, E2 + PTH but not E2 alone, had biphasic effects on ALP activity, with maximum stimulation observed at 10(-11) and 10(-10)M E2, and a return to basal levels at 10(-9)M E2. E2 decreased basal adenylate cyclase activities in a dose-dependent manner in (HOB + DEX) but not (HOB - DEX) cells (KW = 13.48, p < 0.05). In (HOB + DEX) cells, E2 had biphasic effects on PTH-stimulated adenylate cyclase activity, with significant stimulation observed at 10(-10)M (p < 0.05). While E2 had no significant effect on osteoblastic marker mRNA levels in (HOB - DEX) cells, it decreased osteocalcin and stimulated PTHr mRNA levels in (HOB + DEX) cells. Thus, in our human osteoblastic cell models, estrogen regulated metabolic function largely in the more differentiated cells, by modifying the effects of PTH.

Asunto(s)

Dexametasona/farmacología , Estradiol/farmacología , Glucocorticoides/farmacología , Osteoblastos/metabolismo , Hormona Paratiroidea/farmacología , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Análisis de Varianza , Northern Blotting , División Celular/efectos de los fármacos , Células Cultivadas , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Quimioterapia Combinada , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteocalcina/biosíntesis , Osteocalcina/genética , ARN Mensajero/metabolismo , Radioinmunoensayo

RESUMEN

Pseudohypoparathyroidism (PHP) is characterized by a lack of response to parathyroid hormone (PTH); however, normal skeletal responsiveness to PTH in some patients with PHP type Ia was previously suggested on the basis of clinical observations. To test this hypothesis, we measured cyclic adenosine monophosphate (cAMP) production in response to various agonists in bone-derived osteoblast-like (OBL) cells from trabecular explants obtained from an iliac crest biopsy of a 25-year-old woman with PHP. The patient was proved to have PHP type Ia on the basis of Albright's hereditary osteodystrophy and decreased activity of stimulatory guanine nucleotide-binding protein (Gs) in erythrocytes. Responsiveness of the patient's OBL cells was compared with OBL cells from eight subjects aged 18-39 years who had no evidence of metabolic bone disease. OBL cells from the patient responded to the following agonists (expressed in multiples of elevation of cAMP, stimulated/basal, mean +/- SE, n = 3): PTH, 3.8 +/- 0.3; forskolin, 8.2 +/- 0.2; and cholera toxin, 56.8 +/- 10.0. These responses were not significantly different from those of control OBL cells: PTH, 4.5 +/- 1.1 (range 2.4-7.5); forskolin, 7.7 +/- 1.4; and cholera toxin, 57.9 +/- 16.2. The normal cholera toxin response indicated the presence of functional Gs. Bone cells from patients with PHP type Ia may exhibit a normal PTH receptor-coupled adenylyl cyclase system in vitro despite clinical evidence of impaired hormone-responsive adenylyl cyclase in other tissues, including the kidney. Skeletal responsiveness to PTH may explain the long periods of spontaneous normocalcemia observed in this patient.

Asunto(s)

Huesos/efectos de los fármacos , Huesos/metabolismo , Hormona Paratiroidea/farmacología , Seudohipoparatiroidismo/metabolismo , Adenilil Ciclasas/metabolismo , Adolescente , Adulto , Fosfatasa Alcalina/metabolismo , Huesos/patología , Células Cultivadas , AMP Cíclico/biosíntesis , Femenino , Humanos , Masculino , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteocalcina/biosíntesis , Seudohipoparatiroidismo/clasificación , Seudohipoparatiroidismo/patología

RESUMEN

We tested the effect of osteoblastic differentiation on the interactive effects of 17 beta-oestradiol (E2) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on alkaline phosphatase activity. As cell models we utilized the more differentiated human osteosarcoma (SaOS) cells that had been cultured for 6 days in medium containing 10 nM dexamethasone (Dex) (SaOS+Dex cells) and the less differentiated cells cultured in the absence of Dex (SaOS-Dex cells). The cells were challenged with 1,25(OH)2D3 in the presence or absence of Dex for 24 h and then with E2 for an additional 24 h. In SaOS-Dex cells, alkaline phosphatase activity remained constant over the 48-h period and was not significantly affected by E2, 1,25(OH)2D3 or 1,25(OH)2D3+E2 treatment. On the other hand, in SaOS+Dex cells, 1,25(OH)2D3 and E2+1,25(OH)2D3 stimulated alkaline phosphatase activity (ANOVA, F = 154.2, P < 0.0001) with the maximal response at 48 h (P < 0.01). In SaOS+Dex cells, 1,25(OH)2D3 had dose-dependent stimulatory effects which were strongly enhanced by 10 nM E2 (ANOVA, F = 46.0, P < 0.001). Studies on dose-dependent effects of E2, in the presence or absence of 100 nM 1,25(OH)2D3, revealed that in the presence of 1,25(OH)2D3, the E2 dose-response curve was biphasic in SaOS+Dex cells (ANOVA, F = 3.40, P < 0.005), with maximum stimulation at 10 nM E2 (P < 0.01). The specificity of E2 was verified using the inactive 17 alpha-oestradiol and the oestrogen antagonist, tamoxifen. These data indicate that E2 and 1,25(OH)2D3 have positive interactive effects on alkaline phosphatase activity in human osteoblasts, and suggest that the expression of this interaction is dependent on the stage of differentiation of the cells.

Asunto(s)

Fosfatasa Alcalina/metabolismo , Calcitriol/farmacología , Estradiol/farmacología , Osteoblastos/efectos de los fármacos , Osteosarcoma/enzimología , Diferenciación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Osteoblastos/enzimología , Estimulación Química , Tamoxifeno/farmacología , Células Tumorales Cultivadas

RESUMEN

We have previously shown that osteoblasts derived from trabecular bone explants and cultured long term in 10 nM dexamethasone ((HOB + DEX) cells) exhibited properties consistent with a more differentiated phenotype compared with those grown in the absence of dexamethasone ((HOB-DEX) cells). To characterize these two cell models further, we measured the steady-state mRNA levels of the phenotypic markers alkaline phosphatase (ALP), collagen type I (COLL) and osteocalcin (OC), OC production, and the activities of ALP and parathyroid hormone (PTH)-stimulated adenylate cyclase. These findings were then correlated with the age and sex of the bone donors. Long-term culture in dexamethasone significantly increased ALP and OC mRNA levels and the activities of ALP and PTH-stimulated adenylate cyclase but not OC production, in (HOB + DEX) compared with (HOB-DEX) cells (p < 0.05). When the data were examined with respect to the age of the bone donor, age-dependent differences in the expression and responses to dexamethasone were apparent. ALP and PTH-stimulated adenylate cyclase activities decreased with increasing age of the bone donor in (HOB-DEX) and (HOB + DEX) cells (p < 0.05). There were no significant correlations between phenotypic marker mRNA levels and bone donor age in (HOB-DEX) and ((HOB + DEX) cells. All age-dependent decreases in ALP and PTH-stimulated cyclase activities were enhanced in the (HOB + DEX) cells. However, when the data were examined according to the sex of the bone donor, there were no differences in mRNA levels, OC production, or ALP and cyclase activities between cells from male and female donors. These results indicate an age dependence in the expression of osteoblastic markers in human bone cells at different stages of differentiation: thus osteoblastic cultures derived from older donors are likely to contain fewer osteoprogenitor cells, lower levels of glucocorticoid receptors or represent more differentiated osteoblasts compared with those derived from younger donors.

Asunto(s)

Fosfatasa Alcalina/metabolismo , Dexametasona/farmacología , Osteoblastos/metabolismo , Adenilil Ciclasas/metabolismo , Adolescente , Adulto , Factores de Edad , Anciano , División Celular/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Proteínas/metabolismo , ARN Mensajero/análisis

RESUMEN

Previous findings in our laboratory have shown that hPTH-(53-84) stimulates alkaline phosphatase activity in dexamethasone-treated ROS 17/2.8 cells. In the present study, we examined the effects of hPTH-(53-84) and hPTH-(1-34) on the expressions of alkaline phosphatase, osteocalcin, and collagen type I mRNA levels in the human osteosarcoma cell line SaOS-2. The effect of hPTH-(53-84) on alkaline phosphatase and osteocalcin message levels was dose dependent (ANOVA, p < 0.005 and p < 0.001, respectively), with significant stimulation observed at 10 nM. Treatment with 10 nM hPTH-(53-84) for 24 h resulted in significant 2- and 1.4-fold increases in mRNA levels for alkaline phosphatase and osteocalcin, respectively (p < 0.05), but had no effect on collagen type I expression. The increased alkaline phosphatase mRNA levels was associated with a 1.5-fold increase in enzyme activity (p < 0.05). In contrast, under similar incubation conditions, hPTH-(1-34) had no significant effects on alkaline phosphatase or osteocalcin mRNA levels. On the other hand, hPTH-(1-34) had dose-dependent stimulatory effects on collagen type I mRNA levels (ANOVA, p < 0.001), 10 nM hPTH-(1-34) stimulating collagen type I expression 1.6-fold (p < 0.05). The results indicate that carboxyl-terminal hPTH-(53-84) has direct and unique biologic effects in human osteoblast-like cells in culture.

Asunto(s)

Fosfatasa Alcalina/metabolismo , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Hormona Paratiroidea/farmacología , Fragmentos de Péptidos/farmacología , Fosfatasa Alcalina/genética , Línea Celular , Colágeno/genética , Colágeno/metabolismo , Humanos , Osteocalcina/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Teriparatido

RESUMEN

We studied the effects of 17 beta-estradiol (E2) and PTH on alkaline phosphatase activity in SaOS-2 cells that had been passaged and grown continually in the presence (SaOS + Dex) or absence (SaOS - Dex) of 10(-8) M dexamethasone (Dex). We showed that the more differentiated SaOS + Dex cells had higher alkaline phosphatase activity and PTH-responsive adenylate cyclase than the less differentiated SaOS - Dex cells. In SaOS - Dex cells, E2 or PTH (1 x 10(-11)-1 x 10(-6) M) had no effect on alkaline phosphatase activity. On the other hand, in SaOS + Dex cells, PTH and E2 each had small but very significant stimulatory effects on alkaline phosphatase activity. The combined effects of PTH and E2 resulted in potentiation which was dose dependent for PTH and E2. 17 alpha-estradiol and tamoxifen (1 x 10(-11)-1 x 10(-6) M) had no effect on PTH-stimulated alkaline phosphatase activity in SaOS + Dex cells, but the latter inhibited the E2 effect, clearly demonstrating its specificity. In conclusion, we have shown that in more differentiated osteoblastic cells, E2 and PTH have interactive effects on alkaline phosphatase activity.

Asunto(s)

Fosfatasa Alcalina/metabolismo , Diferenciación Celular/fisiología , Estradiol/farmacología , Hormona Paratiroidea/farmacología , Adenilil Ciclasas/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Clonales , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Osteoblastos , Osteosarcoma , Tamoxifeno/farmacología , Células Tumorales Cultivadas

RESUMEN

We have examined bone cells derived from iliac crest trabecular explants of 30 patients with idiopathic osteoporosis and 45 control subjects in order to determine whether intrinsic abnormalities in osteoblast function may contribute to the decreased bone formation observed in this disease. Bone cells isolated from all subjects expressed several in vitro characteristics of the osteoblast phenotype including adenylate cyclase responsiveness to parathyroid hormone (PTH) and prostaglandin E1 (PGE1), basal and 1,25(OH)2D3-stimulated alkaline phosphatase activity and osteocalcin production. Results were compared amongst three subject groups; young controls less than 40 years old, older controls over 40 years old, and osteoporotics. Osteoporotic cells were found in general to be fully active in vitro. There were no differences between osteoporotic and control cells in their basal levels of adenylate cyclase, or alkaline phosphatase, in their growth rates, or cell morphology. The cyclic AMP (cAMP) response to PTH was significantly lower in osteoporotic cells (71%, p < 0.01) and older control cells (64%, p < 0.005) relative to the response in cells from younger controls, suggesting that the decreased responsiveness in osteoporotic cells was due to subject age rather than the osteoporotic state. At the same time, the cAMP responses to PGE1 and cholera toxin were similar in cells from all three subject groups. The response to forskolin was reduced to about 40% in osteoporotic cells compared with controls, but this was not mirrored by similar differences in the responses to PTH, PGE1 or cholera toxin, suggesting that the availability of catalytic subunits is not rate-limiting in these cells. 1,25(OH)2D3-stimulated osteocalcin production was 220% higher in osteoporotics than in older controls, but the numbers tested were small and the difference did not reach significance. The one significant abnormality we observed in osteoporotic cells was in alkaline phosphatase activity: 1,25(OH)2D3-stimulated alkaline phosphatase activity was twofold higher in osteoporotics than in younger (p < 0.05), older (p < 0.05) and pooled controls (p < 0.025). The significance of this finding is unknown, but we postulate that it may reflect an intrinsic abnormality in osteoblast function in patients with idiopathic osteoporosis.

Asunto(s)

Osteoblastos/metabolismo , Osteoblastos/patología , Osteoporosis/metabolismo , Osteoporosis/patología , Adolescente , Adulto , Fosfatasa Alcalina/metabolismo , Calcitriol/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Femenino , Humanos , Masculino , Osteocalcina/metabolismo , Hormona Paratiroidea/farmacología , Valores de Referencia

RESUMEN

We tested whether the protein kinase C (PKC) modulation of PTH-sensitive adenylate cyclase in ROS 17/2.8 cells is affected by the glucocorticoid dexamethasone and the vitamin D hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Basal and PTH- and forskolin-stimulated adenylate cyclase activities were determined in the presence or absence of 100 nM phorbol 12-myristate 13-acetate (PMA), the activator of PKC, in ROS 17/2.8 cells that had been previously cultured with or without dexamethasone or 1,25(OH)2D3. Dexamethasone treatment increased the basal, PMA-, PTH-, (PTH + PMA)- and (forskolin + PMA)-sensitive adenylate cyclase while 1,25(OH)2D3 decreased these effects. The stimulatory and inhibitory effects were dose-dependent with respect to dexamethasone and 1,25(OH)2D3, respectively. Dexamethasone increased, while 1,25(OH)2D3 decreased the maximal activity of both PTH-sensitive and PKC-modulated PTH-sensitive adenylate cyclase without affecting the half-maximal concentration (ED50) of PTH required for the activation of the enzyme. Additionally, dexamethasone, 1,25(OH)2D3 and PKC did not affect each other's ED50. Our results suggest that the effects of dexamethasone, 1,25(OH)2D3 and PKC on PTH-sensitive adenylate cyclase in ROS 17/2.8 cells are independent of each other.

Asunto(s)

Adenilil Ciclasas/metabolismo , Calcitriol/farmacología , Dexametasona/farmacología , Osteoblastos/enzimología , Hormona Paratiroidea/farmacología , Proteína Quinasa C/farmacología , Animales , Colforsina/farmacología , Activación Enzimática/efectos de los fármacos , Cinética , Osteoblastos/efectos de los fármacos , Osteosarcoma , Ratas , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas