Modulation of gene expression in human osteoblasts by targeting a distal promoter region of human estrogen receptor-alpha gene

Abstract

Estrogen receptor (ER) is expressed during osteoblast differentiation; however, both its functional role in bone metabolism and its involvement in osteoporotic pathogenesis caused by estrogen deficiency are not well understood. Loss of ER gene expression could be one of the mechanisms leading to osteoporosis. Therefore, we investigated a possible modulation of ER gene expression in a human osteoblastic cell line and in four primary osteoblast cultures by using a decoy strategy. Double stranded DNA molecules, mimicking a regulatory region of the ER gene promoter (DNA-102) and acting as a 'silencer' in breast cancer cells, were introduced into osteoblasts as 'decoy' cis-elements to bind and functionally inactivate a putative negative transcription factor, and thus to induce ER gene expression.We found that the DNA-102 molecule was able to specifically bind osteoblast nuclear proteins.Before decoy treatment, absence or variable low levels of ER RNAs in the different cultures were detected. When the cells were transfected with the DNA-102 decoy, an increase in expression of ER and osteoblastic markers, such as osteopontin, was observed, indicating a more differentiated osteoblastic phenotype both in the cell line and in primary cultures. These results showed that the DNA-102 sequence competes with endogenous specific negative transcription factors that may be critical for a decrease in or lack of ER gene transcription. Therefore, osteoblastic transfection with the DNA-102 decoy molecule may be considered a tempting model in a putative therapeutic approach for those pathologies, such as osteoporosis, in which the decrease or loss of ER expression plays a critical role in bone function.

Effects of a Hydroxyapatite-based Biomaterial on Gene Expression in Osteoblast-like Cells

“…MG-63 and SaOS-2 osteosarcoma cells were cultured in minimal essential medium (MEM) with 10% fetal bovine serum (FBS), as previously described (Lambertini et al, 2002). Biostite ® was frozen in liquid nitrogen, pulverized in a micro-dismembrator (Braun, Melsungen, Germany), suspended in medium, then added at different concentrations (from 2.5 to 2500 g/mL) and times (from 1 to 6 days) to cells previously seeded in either 24-well plates or flasks.…”

“…Electrophoretic bands were analyzed by a densitometer (Bio-Rad Laboratories S.r.l., Segrate, Mi, Italy). Alkaline phosphatase activity (ALP) was measured in cultured cells by hydrolysis of p-nitrophenylphosphate and expressed as U/mg protein, as previously described (Lambertini et al, 2002).…”

Hypomethylation of Alu Elements in Post-Menopausal Women with Osteoporosis

“…DNA methylation is a possible mechanism of epigenetic regulation of bone development and osteoporosis and plays a significant role in osteogenic cell differentiation and bone metabolism [20]–[22]. Alu is the most abundant of the short-interspersed nuclear elements (SINE), with over one million copies per genome.…”

Methylation analysis of the promoter F of estrogen receptor α gene: effects on the level of transcription on human osteoblastic cells

“…Bone samples were collected during surgery following Hardinge's surgical approach to the hip and the bone was cultured as previously described [15]. Briefly, bone was cut into small pieces that were rinsed and then cultured in Eagle's minimum essential medium (Sigma Aldrich, Chemical Co., St. Louis, MO, USA) supplemented with 20% fetal bovine serum (Invitrogen Corporation, CA, USA), 2 mM glutamine, 100 U/ml penicillin, 100 g/ml streptomycin and 50 g/ml ascorbate, at 37 • C in an humidified atmosphere of 5% CO 2 .…”