Age and gender specific stimulation of creatine kinase specific activity by gonadal steroids in human bone-derived cells in culture

Abstract

We previously reported a non-enzymatic method for isolation of human bone cells in culture that display osteoblastic features and respond to 1,25 dihydroxy vitamin D (1,25) and to parathyroid hormone (PTH). The present study was undertaken to analyze the response of cultured human bone cells to 17beta-estradiol (E2) and to dihydrotestosterone (DHT) as a function of gender and age. Cultured human bone cells, obtained from biopsies during orthopedic surgery, were divided into four groups defined by gender and age: pre- and post-menopausal healthy non-osteoporotic women that were not under hormone replacement therapy (HRT) and mature (<55-year-old) and older (>60-year-old) men. We found gender specific responses to gonadal steroids using the specific activity of the brain type (BB) isozyme of creatine kinase (CK) as a response marker. Constitutive levels of CK activity did not change with age or gender and the enzyme extracted from cells from the different sexes and ages did not respond to either progesterone (P) or to 1,25. CK from the different cells responded to gonadal steroids in a gender specific manner, i.e. CK from female derived cells responded to E2 only and the enzyme from male derived cells responded to DHT only. In female derived cells the response to E2 declined significantly with age, while the response to DHT in CK from male derived cells did not vary with age. This may be due to either decreased proportion of mature osteoblasts and/or their differentiation state and/or changes in the levels of estrogen receptor(s), coactivators or corepressors in these cells. These results extend our knowledge of human osteoblast biology (beyond murine cells) and are therefore more relevant for developing models for treatment of human metabolic bone diseases such as post-menopausal osteoporosis.

Runx2 Integrates Estrogen Activity in Osteoblasts

“…1B). Therefore, these easily obtained and well characterized osteoblast cultures can be made estrogen responsive in vitro, replicating the hormone-sensitive status of osteoblasts that occur in more mature organisms in vivo (43)(44)(45)(46). Importantly, this osteoblast cell culture model provides a sensitive system to examine specific aspects of ER␣ on osteoblast activity uncomplicated by the presence of endogenous ERs.…”

“…We chose to avoid possible complications from antibiotic toxicity, unknown effects from stable gene integration, and phenotypic drift by cells in continuous culture with our approach. Even so, it is difficult to know how the level of ER␣ expression in any transfected cell model compares with that in adult bone because, even in vivo, levels of ER␣ vary considerably with age and with anatomical bone location (43)(44)(45)(46). However, expression of ER␣ by transient gene transfection seemed appropriate and predictable, because the ability of estrogen to driven reporter gene expression by ERE was steroid- FIG.…”

Phytoestrogenic Compounds and Their Synthetic Analogs, Contrary to Estradiol- 17b Stimulates Human Derived Female Cultured Bone Cells in Hyperglycemic Conditions

“…Cells were scraped off the culture dishes and homogenized by freezing and thawing three times in cold isotonic extraction buffer [12]. Supernatant extracts were obtained by centrifugation at 14000xg for 5 min at 4°C in an Eppendorf micro-centrifuge.…”

“…In human-derived cultured osteoblasts (hObs), we found that E 2 increased cell proliferation and CK specific activity in a gender specific manner [12] as a response marker for hormonal treatment beyond estrogen itself in cells containing the relevant receptors.…”

“…Creatine kinase specific activity (CK) was measured in a Kontron Model 922 Uvicon Spectrophotometer at 340nm using a Sigma coupled assay kit (procedure 47-UV). Protein was assayed by Coomassie brilliant blue dye binding, using BSA as the standard [12]. …”