In the present study, we investigated the role of the phytoestrogen genistein and 17beta-estradiol in human bone marrow stromal cells, undergoing induced osteogenic or adipogenic differentiation. Profiling of estrogen receptors (ERs)-alpha, -beta1, -beta2, -beta3, -beta4, -beta5, and aromatase mRNAs revealed lineage-dependent expression patterns. During osteogenic differentiation, the osteoblast-determining core binding factor-alpha1 showed a progressive increase, whereas the adipogenic regulator peroxisome proliferator-activated receptor gamma (PPARgamma) was sequentially decreased. This temporal regulation of lineage-determining marker genes was strongly enhanced by genistein during the early osteogenic phase. Moreover, genistein increased alkaline phosphatase mRNA levels and activity, the osteoprotegerin:receptor activator of nuclear factor-kappaB ligand gene expression ratio, and the expression of TGFbeta1. During adipogenic differentiation, down-regulation in the mRNA levels of PPARgamma and CCAAT/enhancer-binding protein-alpha at d 3 and decreased lipoprotein lipase and adipsin mRNA levels at d 21 were observed after genistein treatment. This led to a lower number of adipocytes and a reduction in the size of their lipid droplets. At d 3 of adipogenesis, TGFbeta1 was strongly up-regulated by genistein in an ER-dependent manner. Blocking the TGFbeta1 pathway abolished the effects of genistein on PPARgamma protein levels and led to a reduction in the proliferation rate of precursor cells. Overall, genistein enhanced the commitment and differentiation of bone marrow stromal cells to the osteoblast lineage but did not influence the late osteogenic maturation markers. Adipogenic differentiation and maturation, on the other hand, were reduced by genistein (and 17beta-estradiol) via an ER-dependent mechanism involving autocrine or paracrine TGFbeta1 signaling.

Estrogene has a tonicising effect upon urethra musculature. There is also the same demonstrable influence of α-receptor stimulating drugs upon the urethra pressure profile. After preliminary treatment with estrogene, there is a considerably stronger tonicising effect of α-stimulating drugs than without such a treatment. So the combination of estrogene and α-stimulating drugs seems a useful addition to the conservative treatment of stress incontinence.

Introduction We report on the in vitro and ex vivo effects of chiral (R)‐10‐hydroxystearic acid (10‐HSA) compared with other mono‐hydroxystearic acid regioisomers and stearic acid (SA) together with its benefit when combined with retinol. Methods Following treatment with hydroxystearic acids peroxisomal proliferator‐activated receptor alpha (PPARα) activity was determined in a luciferase reporter gene assay, collagen type I was assessed in primary human dermal fibroblasts by immunohistochemistry, modification of the intracellular fibroblast collagen proteome was studied by mass‐spectrometry‐based proteomics and collagen type III was assessed by immunohistochemistry on human ex vivo skin. Results 10‐HSA was the most effective PPARα agonist (15.7× induction; p < 0.001), followed by 9‐HSA (10.1× induction) and then 12‐HSA (4.9× induction) with 17‐HSA (1.7× induction) being similar to the effects of stearic acid (1.8× induction). Collagen type I levels were increased in primary human fibroblasts by 2.12× and 1.56× for 10‐HSA and 9‐HSA, respectively, in vitro with the10‐HSA being significant (p < 0.05), whereas 12‐HSA and SA had no statistical effect over the untreated control. 10‐HSA and 12‐HSA modified the intracellular fibroblast collagen proteome slightly with significant increases in collagen alpha‐1 (VI) and alpha‐3 (VI) proteins but only 10‐HSA increased levels of collagen alpha‐2 (V), alpha‐1 (III), alpha‐1 (I) and alpha‐2 (I) (all p < 0.05) with the increases being significantly different between 10‐HSA and 12‐HSA for collagen alpha‐1 (I), collagen‐3 (VI) and collagen alpha‐2 (I) (p < 0.01). Collagen type III in ex vivo skin was increased +47% (p < 0.05) by 0.05% (1.7 mM) retinol, +70% (p < 0.01) by 0.01% (0.33 mM) 10‐HSA and the combination increased levels by +240% (p < 0.01 for either ingredient). Conclusion Chiral (R)‐10‐HSA has been shown to be superior to 9, 12 and 17‐HSA as a PPARα agonist. Moreover, 10‐HSA stimulated collagen synthesis in monolayer fibroblast culture as assessed by proteomics and immunohistochemically. Furthermore, we also show the synergistic effects of 10‐HSA with retinol on collagen III synthesis in skin explants. These results further highlight the efficacy of 10‐HSA as a cosmetically acceptable PPARα agonist and anti‐ageing ingredient.

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Estrogen receptors, members of the nuclear hormone receptor family, are not only able to bind their endogenous hormone, 17beta-estradiol, but can also accommodate other naturally-occuring, non-steroidal molecules. Here, we describe a spin-column procedure to determine accurately equilibrium dissociation constants (Kds) and IC50 concentrations for estrogenic compounds. The human wild-type ERalpha was used to validate the protocol. We expressed the full-length ERalpha protein in an eukaryotic system to ensure all possible post-transcriptional modifications. The gel filtration-based assay revealed a temperature-dependent Kd shift for ERalpha. At physiological conditions (150 mM salt, 37 degrees C) we determined the 17beta-estradiol Kd for ERalpha to be 281 +/- 13 pmol/L. Positive cooperativity was only apparent at low temperatures and diminished to zero at 37 degrees C. In homologous competition binding experiments using 17beta-estradiol, we observed fifty fold higher IC50 values than the respective Kd. This paper presents a reliable and sensitive protocol to generate saturation binding curves and heterologous competition curves to test estrogenic compounds.