Differential Effects of Short Term Feeding of a Soy Protein Isolate Diet and Estrogen Treatment on Bone in the Pre-Pubertal Rat

BACKGROUND: Previous reports suggest that beneficial effects of soy on bone quality are due to the estrogenic actions of isoflavone phytochemicals associated with the protein. However, mechanistic studies comparing the effects of soy diet and estrogens on bone, particularly in rapidly growing animals are lacking. METHODOLOGY AND PRINCIPAL FINDINGS: We studied the effects of short term feeding of soy protein isolate (SPI) on bone in comparison to the effects of 17β-estradiol (E2) in pre-pubertal rats. Female rats were weaned to one of 4 treatments: 1) a control casein-based diet (CAS); 2) CAS with subcutaneous E2 (10 µg/kg/d) (CAS+E2); 3) a SPI-containing diet (SPI); or 4) SPI with subcutaneous E2 (SPI) or SPI with 10 µg/kg/d E2 (SPI+E2) for 14 days beginning on postnatal day 20. SPI increased while E2 decreased bone turnover compared to CAS. In contrast, both treatments decreased serum sclerostin levels. Microarray analysis of RNA isolated from bone revealed 652 genes regulated by SPI, 491 genes regulated by E2, and 266 genes regulated by both SPI diet and E2 compared to CAS. The expression of caveolin-1, a protein localized in the cell membrane, was down-regulated (p<0.05) in rats fed SPI, but not by E2 compared to rats fed casein. Down-regulation of caveolin-1 by SPI was associated with increased BMP2, Smad and Runx2 expression in bone and osteoblasts (p<0.05). CONCLUSIONS/SIGNIFICANCE: These results suggest SPI and E2 have different effects on bone turnover prior to puberty. Approximately half of the genes are regulated in the same direction by E2 or SPI, but in combination, SPI blocks the estrogen effects and returns the profile towards control levels. In addition, there are E2 specific and SPI-specific gene changes related to regulation of bone formation

Caveolin-1, membrane versus cytoplasm BMP-2 protein expression in bone of pre-pubertal female rats from Control, standard casein diet group; E2, 10 µg/kg/d E2 treated group; SPI, soy protein isolate diet group; E2+SPI, combination of 10 µg/kg/d E2 treated and SPI diet group.

<p>Quantitation of the intensity of the caveolin-1 bands in the autoradiograms was performed relative to expression of β-actin, membrane BMP-2 (M-BMP-2) and nuclear BMP-2 (N-BMP-2) were performed relative to total BMP-2 (T-BMP-2). Data are Means ± S.E.M, n = 3, with different letters differ significantly from each other at p<0.05, a</p

Serum isoflavone concentration.

<p>Serum isoflavones were measured using LC-MS method.</p><p>nd, non detected. DHD, dihydrodaidzein. o-DMA, o-desmethylangolansin.</p

Microarray analysis shows differential signatures of SPI, E2, and SPI+E2 on gene expression in bone in pre-pubertal female rats.

<p>(A) Venn diagrams on the differentially expressed genes among groups. (B) Different pattern of gene regulation by SPI, E2 and SPI+E2 from 266 genes showed in (A). (C) Top 25 genes up-regulated by SPI and all genes down-regulated by SPI from 266 genes. (D) Top 25 genes up-regulated by E2 and all genes down-regulated by E2 from 266 genes showed in (B).</p

Real-time PCR verification of expression of genes of caveolin-1, ATF-3, calcitonin and Sost (sclerostin) in bone.

<p>Black bars represent fold changes with microarray analyses and gray bars represent relative mRNA expression from real-time PCR analyses. Control, standard casein diet group; E2, 10 µg/kg/d E2 treated group; SPI, soy protein isolate diet group; E2+SPI, combination of 10 µg/kg/d E2 treated and SPI diet group. Means ± S.E.M of real-time PCR results with different letters differ significantly from each other at p<0.05, a</p

SPI stimulates osteoblast differentiation via down-regulation of caveolin-1.

<p>ST2 cells were treated with 2% serum either from control, 10 µg/kg/d E2 treated, SPI diet rats and 5 µM of purified genistein for 48 h. RNA and protein were collected. (A), Real-time PCR showing relative caveolin-1 and Runx2 mRNA expression. Data are Means ± SEM, triplicates, * p<0.05 versus control. (B), Western blotting showing caveolin-1 protein, nuclear SMD and cytosolic versus membrane BMP2 expression. Quantitation of the intensity of the caveolin-1 in the autoradiograms was performed relative to expression of β-actin.</p

Cell signaling transduction pathway after activation of BMP-2 by SPI and E2 in bone.

<p>(A), Protein was isolated from bone after aspiration of bone marrow cells and performed Western blots. Smad and ERK1/2 phosphorylation and Runx2 protein expression in pre-pubertal female rat bone from Control, standard casein diet group; E2, 10 µg/kg/d E2 treated group; SPI, soy protein isolate diet group; E2+SPI, combination of 10 µg/kg/d E2 treated and SPI diet group. Quantitation of the intensity of the phosphorylated bands of p-Smad1, 5, 8 and p-ERK1/2 in the autoradiograms were performed relative to expression of total Smad4 and ERK1/2. Blot of Ponceau S staining showing protein loading control for western blotting. (B), Showing real-time PCR analyses for Runx2. Data are Means ± S.E.M, n = 3, with different letters differ significantly from each other at p<0.05, a</p