Bioconversion of norethisterone, a progesterone receptor agonist into estrogen receptor agonists in osteoblastic cells

Abstract

A number of clinical studies have demonstrated that norethisterone (NET), a potent synthetic progestin, restores postmenopausal bone loss, although its mode of action on bone cells is not fully understood, while the effect of naturally occurring progesterone in bone has remained controversial. A recent report claims that the potent effects of NET on osteoblastic cell proliferation and differentiation, mimicking the action of estrogens, are mediated by non-phenolic NET derivatives. To determine whether osteoblasts possess the enzymes required to bioconvert a progesterone receptor (PR) agonist into A-ring reduced metabolites with affinity to bind estrogen receptor (ER), we studied the in vitro metabolism of [ 3 H]-labeled NET in cultured neonatal rat osteoblasts and the interaction of its metabolic conversion products with cytosolic -osteoblast ER, employing a competition analysis. Results indicated that NET was extensively bioconverted (36 . 4%) to 5a-reduced metabolites, including 5a-dihydro NET, 3a,5a-tetrahydro NET (3a,5a-NET) and 3b,5a-tetrahydro NET (3b,5a-NET), demonstrating the activities of 5a-steroid reductase and two enzymes of the aldo-keto reductases family. Expression of Srd5a1 in neonatal osteoblast was well demonstrated, whereas Srd5a2 expression was not detected. The most striking finding was that 3b,5a-NET and 3a,5a-NET were efficient competitors of [ 3 H]-estradiol for osteoblast ER binding sites, exhibiting affinities similar to that of estradiol. The results support the concept that the interplay of 5a-steroid reductase and aldo-keto reductases in osteoblastic cells, acting as an intracrine modulator system is capable to bioconvert a PR agonist into ER agonists, offering an explanation of the molecular mechanisms NET uses to enhance osteoblastic cell activities.

Neonatal rat osteoblasts bioconvert testosterone to non-phenolic metabolites with estrogen-like effects on bone cell proliferation and differentiation

“…Meanwhile, an almost complete absence of diols was noticed when radiolabeled T was incubated with rat osteoblasts in the presence of Flu, an inhibitor of 3 α -and 3 β -hydroxysteroid dehydrogenases. These findings are in line with previous reports from our group, demonstrating that norethisterone (NET), a synthetic progestin derived from 19-nortestosterone, was bioconverted in neonatal rat osteoblasts to the A-ring-reduced metabolites 5 α -dihydro-NET and 3 α -and 3 β -tetrahydro-NET [ 40 ].…”

“…cDNA samples were subjected to quantitative amplifi cation using the TaqMan probe and primers set of Alp 1 (Rn01516026_ml) as previously described [ 40 ]. Gene expression was normalized against β -actin used as a housekeeping gene.…”

The anti-estrogenic activity of indole-3-carbinol in neonatal rat osteoblasts is associated with the estrogen receptor antagonist 2-hydroxyestradiol

“…The relative binding affinity of 2-OHE 1 and 2-OHE 2 to cytosolic ER was assessed using a competition analysis, as previously described by our group [31], using progesterone (P) and E 2 as the controls. Cytosol aliquots (200 µg protein/ml) were incubated with 1 nM [ 3 H]-E 2 at 4 °C for 18 h in the absence or presence of increasing concentrations (1, 5, 10, 100 and 250 nM) of the radio-inert steroids E 2 , P, 2-OHE 1 and 2-OHE 2 .…”

“…Similarly, progesterone, which was used as the negative control, was unable to compete for binding to ER. The inhibition constant (K i ) was calculated for each competitor using K d = 1.19 × 10 −9 M, as previously described in a study by our group that was aimed at characterizing the kinetic constants, at equilibrium, of ER using [ 3 H]-E 2 [31]. The RBA and K i values of each steroid competitor are listed in Table 1.…”

Hormone therapy and breast cancer: emerging steroid receptor mechanisms

“…Whether progestins bind to the ER is contradictory. Some studies suggest that medroxyprogesterone acetate (MPA) and norethisterone (NET) can bind to the ER and elicit estrogenic activity, others suggest that they do not (Larrea et al 2001, Pasapera et al 2002, Escande et al 2006, Lemus et al 2009. Interestingly, we recently showed that NET-acetate (NET-A), levonorgestrel (LNG) and gestodene (GES) can bind to ER-A, but not ER-B, while P 4 , MPA, nestorone (NES), nomegestrol acetate (NoMAC) and drospirenone (DRSP) do not bind to either ER subtype (Louw-du Toit et al 2017).…”