Membranal effects of phytoestrogens and carboxy derivatives of phytoestrogens on human vascular and bone cells: new insights based on studies with carboxy-biochanin A.
Estradiol-17beta (E2) and some phytoestrogens induce a biphasic effect on DNA synthesis in cultured human vascular smooth muscle cells (VSMC), i.e., stimulation at low concentrations and inhibition at high concentrations. These compounds also increase the specific activity of creatine kinase (CK) as well as intracellular Ca2+ concentration in both VSMC and human female-derived cultured bone cells (OBs), and stimulate ERK1/2 phosphorylation in VSMC. At least some of these effects are exerted via membranal binding sites (mER), as would appear from observations that protein-bound, membrane impermeant estrogenic complexes can mimic the effect of E2 on DNA synthesis, intracellular Ca2+ concentration and MAPK, but not on CK activity. We now extend these studies by examining the effects of a novel carboxy-derivative of biochanin A, 6-carboxy-biochanin A (cBA) in VSMC and human osteoblasts in culture. cBA increased DNA synthesis in VSMC in a dose-dependent manner and was able to maintain this effect when linked to a cell membrane impermeable protein. In VSMC both cBA and estradiol, in their free or protein-bound forms induced a steep and immediate rise in intracellular calcium. Both the free and protein-bound conjugates of cBA and estradiol increased net MAPK-kinase activity. Neither the stimulatory effect of cBA nor the inhibitory effect of estradiol on DNA synthesis in VSMC could be shown in the presence of the MAPK-kinase inhibitor UO126. The presence of membrane binding sites for both estradiol and cBA was supported by direct visualization, using fluorescence labeling of their respective protein conjugates, E2-BSA and cBA-ovalbumin. Furthermore, these presumed membrane ER for estradiol and cBA were co-localized. In cultured human osteoblasts, cBA stimulated CK activity in a dose related fashion, which paralleled the increase in CK induced by estradiol per se, confirming the estrogenic properties of cBA in human bone cells. Both the free and protein-bound forms of cBA elicited immediate and substantial increments in intracellular Ca2+, similar to, but usually larger than the responses elicited by estradiol per se. cBA also increased ERalpha and suppressed ERbeta mRNA expression in human osteoblasts. Cultured human osteoblasts also harbor membrane binding sites for protein-bound form of cG, which are co-localized with the binding sites for protein-bound estradiol. The extent to which these properties of the novel synthetic phytoestrogen derivatives may be utilized to avert human vascular and/or bone disease requires further study.
Somjen
D
D
Institute of Endocrinology, Metabolism and Hypertension, Tel Aviv Sourasky Medical Centre and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 64239, Israel. dalias@tasmc.health.gov.il
Kohen
F
F
Lieberherr
M
M
Gayer
B
B
Schejter
E
E
Katzburg
S
S
Limor
R
R
Sharon
O
O
Knoll
E
E
Posner
G H
GH
Kaye
A M
AM
Stern
N
N
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D047628:Estrogen Receptor alpha / Q000378:metabolism; D047629:Estrogen Receptor beta / Q000378:metabolism; D005260:Female; D019833:Genistein / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D048789:Phytoestrogens / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
C462199:6-carboxymethyl genistein; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D048789:Phytoestrogens; D004247:DNA; D019833:Genistein; D003402:Creatine Kinase; D002118:Calcium; C004541:biochanin A