Characterization of 11beta-hydroxysteroid dehydrogenase activity and corticosteroid receptor expression in human osteosarcoma cell lines

Abstract

Studies in vitro and in vivo have shown that corticosteroids play an important role in bone physiology and pathophysiology. It is now established that corticosteroid hormone action is regulated, in part, at the pre-receptor level through the expression of isozymes of 11 -hydroxysteroid dehydrogenase (11 -HSD), which are responsible for the interconversion of hormonally active cortisol to cortisone.In this report we demonstrate 11 -HSD activity in human osteoblast (OB) cells. Osteosarcoma-derived OB cell lines TE-85, MG-63 and SaOS-2 and fibrosarcoma Hs913T cells express the type 2 isoform of 11 -HSD, as determined by reverse transcription polymerase chain reaction (RT-PCR) and specific enzyme assays. Enzyme activity was shown to be strictly NAD dependent with a K m of approximately 71 nM; 11 -HSD type 1 mRNA expression and enzyme activity were not detected. All four cell lines expressed mRNA for the glucocorticoid receptor (GR) and mineralocorticoid receptor, but specific binding was only detectable with radiolabelled dexamethasone (K d =10 nM) and not aldosterone. MG-63 cells had two to three times more GR than the other OB cells, which correlated with the higher levels of 11 -HSD 2 activity in these cells. In contrast to the osteosarcoma cell studies, RT-PCR analysis of primary cultures of human OB cells revealed the presence of mRNA for 11 -HSD 1 as well as 11 -HSD 2. However, enzyme activity in these cells remained predominantly oxidative, i.e. inactivation of cortisol to cortisone (147 pmol/h per mg protein at 500 nM cortisol) was greater than cortisone to cortisol (10·3 pmol/h per mg protein at 250 nM cortisone). Data from normal human OB and osteosarcoma cells demonstrate the presence of an endogenous mechanism for inactivation of glucocorticoids in OB cells. We postulate that expression of the type 1 and type 2 isoforms of 11 -HSD in human bone plays an important role in normal bone homeostasis, and may be implicated in the pathogenesis of steroid-induced osteoporosis.

Expression of 11β-hydroxysteroid dehydrogenase enzymes in human osteosarcoma: potential role in pathogenesis and as targets for treatments

“…Cells were treated with 100 nM DHD or 100 nM DEX with the equivalent tritiated tracers for 6 h for the assessment of enzyme activity. Similar experiments were carried out in the MG-63 OS cell line, which has previously been shown to have high levels of 11b-HSD2 expression (Bland et al 1999). All experiments were carried out in triplicate on three separate occasions.…”

“…The 11b-HSD1 enzyme metabolises endogenous glucocorticoids, cortisone and cortisol, in a bidirectional manner, whereas 11b-HSD2 is a unidirectional inactivator of endogenous glucocorticoids. We have previously demonstrated that OS cell lines express the 11b-HSD2 enzyme (Bland et al 1999, Eyre et al 2001. This is in contrast to normal osteoblasts that express the 11b-HSD1 enzyme (Cooper et al 2000.…”

Interactions between GH, IGF-I, Glucocorticoids, and Thyroid Hormones during Skeletal Growth

“…The type 2 enzyme, 11␤HSD2, is a dehydrogenase that catalyzes inactivation of GC to protect the nonselective mineralocorticoid receptor from GC activation in target tissues such as the kidney, or to prevent the passage of maternal GC across the placenta and protect the fetus (104). Thus, local tissue GC concentrations are modulated by the 11␤HSD2 enzyme, and 11␤HSD2 activity has been demonstrated in osteoclasts and osteoblasts (105,106), although its function in bone is unknown (107). If 11␤HSD enzymes are expressed in growth plate chondrocytes, they may act as significant GH-and IGF-I-sensitive regulators of local GC concentrations in the growth plate.…”

Glucocorticoid activity, inactivity and the osteoblast

“…In addition to endogenous glucocorticoids, the synthetic glucocorticoids, prednisolone and dexamethasone, are substrates for 11 -HSD, though with reduced affinity compared with cortisol and cortisone. We have recently demonstrated the presence of 11 -HSDs in human osteoblasts: 11 -HSD1 in osteoblast primary cultures (Bland et al 1999), cell lines and normal adult bone (Cooper et al 1998); 11 -HSD2 in osteosarcoma cell lines (Bland et al 1999) and normal fetal bone (Condon et al 1998). 11 -HSD expression and activity are regulated by factors likely to be present in the bone microenvironment Figure 2 Schematic illustration of the role of 11 -HSD enzymes in the osteoblast.…”