Abstract
Mechanical stimulation induces bone growth and remodeling by the secondary messenger, cyclic guanosine 3’, 5’-monophosphate (cGMP), in osteoblasts. However, the role of cGMP in the regulation of estrogen biosynthesis, whose deficiency is a major cause of osteoporosis, remains unclear. Here, we found that the prenylated flavonoids, 3-O-methoxymethyl-7-O-benzylicaritin (13), 7-O-benzylicaritin (14), and 4'-O-methyl-8-isopentylkaempferol (15), which were synthesized using icariin analogs, promoted estrogen biosynthesis in osteoblastic UMR106 cells, with calculated EC50 values of 1.53, 3.45, and 10.57 µM, respectively. 14 and 15 increased the expression level of the bone specific promoter I.4-driven aromatase, the only enzyme that catalyzes estrogen formation by using androgens as substrates, in osteoblastic cells. 14 inhibited phosphodiesterase 5 (PDE5), stimulated intracellular cGMP level and promoted osteoblast cell differentiation. Inhibition of cGMP dependent-protein kinase G (PKG) abolished the stimulatory effect of 14 on estrogen biosynthesis and osteoblast cell differentiation. Further, PKG activation by 14 stimulated the activity of SHP2 (Src homology 2 domain-containing tyrosine phosphatase 2), thereby activating Src and ERK (extracellular signal-regulated kinase) signaling and increasing ERK-dependent aromatase expression in osteoblasts. Our findings reveal a previously unknown role of cGMP in the regulation of estrogen biosynthesis in the bone. These results support the further development of 14 as a PKG-activating drug to mimic the anabolic effects of mechanical stimulation of bone in the treatment of osteoporosis.
Highlights
Osteoporosis is a major global public health problem caused by the reduced estrogen level in postmenopausal women [1]
We showed that the phosphodiesterase 5 (PDE5) inhibitors, sildenafil and icariin analogs, promote aromatase expression in human ovarian granulosalike KGN cells by activating the cAMP/CREB pathway [13]
To search for small molecules that modulate estrogen biosynthesis, we examined the effects of icariin analogs (16, 17, Figure S1) and their effects on estrogen biosynthesis in the rat osteoblast-like cell line, UMR106
Summary
Osteoporosis is a major global public health problem caused by the reduced estrogen level in postmenopausal women [1]. The aromatase expression at various sites is regulated by tissue-specific promoters through the alternative splicing mechanisms [3]. Class I cytokines such as TGF-b1, IL-1b, and TNF-a drive aromatase expression by the usage of promoter I.4 [4]. Aromatase activity is a key factor in skeletal development and mineralization, and is crucial to estrogen production in the bone [5]. Aromatase activity may decline with an increase in during aging, and the contribution of such decline to age-related bone loss is similar in magnitude to that of sex steroid deficiency in both women and men [6, 7]. Agonists of aromatase expression or activity in the bone would be a new therapeutic means for preventing and treating osteoporosis
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