Estrogen enhances α_vβ₃ integrin expression by avian osteoclast precursors via stabilization of β₃ integrin mRNA

Although bone resorption is accelerated with menopause, the means by which diminished circulating 17β-estradiol (E₂) promotes osteoclastic activity are unknown. We hypothesized that since the integrin α_vβ₃ is essential to the resorptive process, reduced E₂ levels may increase the integrin's expression by osteoclast precursors. Thus, avian osteoclast precursors (known to contain E₂ receptors) were exposed ± E₂, surface iodinated, and lysed. The lysate was immunoprecipitated with an antibody recognizing the intact α_vβ₃ heterodimer. We find E₂ alone fails to impact on α_vβ₃ expression. Most importantly, however, picomolar (i.e. post-menopausal), but not nanomolar (i.e. premenopausal) concentrations of E₂, when added in conjunction with 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃], enhance α_vβ₃ expression on the plasma membrane of avian osteoclast precursors relative to 1,25-(OH)₂D₃ alone. Induction of α_vβ₃ by picomolar levels of E₂ is dose-dependent, maximizing at 10^-11-10^-12 M, wherein the sex steroid enhances 1,25-(OH)₂-D₃-stimulated integrin expression approximately 2.5-fold. Northern analysis reveals that β₃ mRNA levels parallel those of α_V3β₃. E₂ (10^-12 M) increases expression of β₃ mRNA induced by a range of 1,25-(OH)₂D₃ concentrations extending from 10^-10 M-10^-8 M. The E₂ + 1,25-(OH)₂D₃ additive effect on β₃ mRNA appears as early as 1 day of treatment and progresses for at least 3 days. Consistent with evidence that the β₃ subunit regulates heterodimer expression, the sex steroid does not impact α_v mRNA. Attesting to the specificity of E₂ on β₃ mRNA expression, the steroid does not impact on β₅ mRNA, and its stereoisomer, 17αE₂, is inactive in these experiments. Likewise, E₂ has no effect on retinoic acid-induced stimulation of β₃ mRNA levels. While 1,25-(OH)₂D₃ induction of β₃ mRNA reflects transcriptional activation, nuclear run-on studies indicate that, despite its inductive effect on β₃ mRNA levels, E₂ does not alter β₃ gene transcription. Transcriptional arrest experiments demonstrate the t1/2 of β₃ mRNA derived from 1,25-(OH)₂D₃-treated cells cultured in the presence or absence of 10^-8 M E₂ is approximately 4 h. On the other hand, 10^-12 M E₂, in conjunction with 1,25-(OH)₂D₃, more than triples stability of β₃ message. Thus, in conjunction with 1,25-(OH)₂D₃, E₂, at levels circulating in postmenopausal (but not premenopausal) females, in whom the rate of bone resorption is accelerated, up-regulates, post-transcriptionally, in osteoclast precursors, α_vβ₃, an integrin heterodimer pivotal to the resorptive process.