Volker Viereck

Differential regulation of Cbfa1/Runx2 and osteocalcin gene expression by vitamin-D3, dexamethasone, and local growth factors in primary human osteoblasts†

Core binding factor alpha 1 (Cbfa1) is an osteoblast‐specific transcription factor essential to develop a mature osteoblast phenotype. However, its exact role in the signaling of various osteotropic‐differentiating agents is still unclear. In this study, we assessed the effects of 1,25‐(OH)2‐D3 (D3), ascorbic acid, bone morphogenetic protein‐2 (BMP‐2), dexamethasone (Dex), and transforming growth factor‐β (TGF‐β) on Cbfa1 and osteocalcin (OCN) mRNA steady state levels (by semiquantitative RT‐PCR) in an in vitro model of osteoblast differentiation. TGF‐β increased Cbfa1 mRNA levels in normal primary human osteoblasts (pHOB) by 2.6‐fold in a time‐dependent fashion with maximum effect on day 28 (P < 0.001). Similarly, the glucocorticoid Dex enhanced Cbfa1 gene expression by pHOB in a time‐dependent fashion by up to 4.6‐fold (P < 0.001). In contrast, Dex inhibited OCN gene expression levels by 68% (P < 0.01). Treatment with BMP‐2 resulted in an earlier enhancement of Cbfa1 and led to a 4.2‐fold increase with a maximum on day 21 (P < 0.001). Ascorbic acid did not modulate Cbfa1 and OCN gene expression. The effect of vitamin D (D3) on Cbfa1 mRNA expression was influenced by the duration of treatment, being inhibitory after 1 h and having a stimulatory effect after 48 h. Time course experiments indicated a stimulatory effect of D3 on Cbfa1 mRNA levels (by 2.5‐fold after 48 h; P < 0.01). Analysis of the late cellular differentiation marker osteocalcin revealed that D3 increased OCN gene expression by 14‐fold (P < 0.001). In conclusion, in normal primary human osteoblasts, the rapid and pronounced increase of OCN after treatment with D3 seems not to be mediated by Cbfa1. These data imply that Cbfa1 gene expression is differentially regulated by various osteoblastic differentiating agents and is dependent on the stage of maturation. J. Cell. Biochem. 86: 348–356, 2002.

Updated recommendations on ultrasonography in urogynecology

Ultrasound is a supplementary, indispensable diagnostic procedure in urogynecology; perineal, introital, and endoanal ultrasound are the most recommended techniques. The position and mobility of the bladder neck can be demonstrated. In patients undergoing diagnostic work-up for urge symptoms, ultrasound occasionally demonstrates urethral diverticula, leiomyomas, and cysts in the vaginal wall. These findings will lead to further diagnostic assessment. The same applies to the demonstration of bladder diverticula, foreign bodies in the bladder, and bullous edema. With endoanal ultrasound, different parts of the sphincter ani muscle can be evaluated. Recommendations for the standardized use of urogenital ultrasound are given.

Phytoestrogen genistein stimulates the production of osteoprotegerin by human trabecular osteoblasts.

The anti‐resorptive effects of estrogen on bone metabolism are thought to be mediated through modulation of paracrine factors produced by osteoblastic lineage cells that act on osteoclastic lineage cells. Receptor activator of nuclear factor‐κB ligand (RANKL) is the essential factor for osteoclast formation and activation and enhances bone resorption. By contrast, osteoprotegerin (OPG), which is produced by osteoblastic lineage cells acts as a decoy receptor that neutralizes RANKL and prevents bone loss. Recently, 17β‐estradiol was found to stimulate OPG mRNA levels and protein secretion in a human osteoblastic cell line through activation of the estrogen receptor (ER)‐α. In this study, we assessed the effects of the phytoestrogen genistein on OPG mRNA steady state levels (by semiquantitative RT‐PCR and Northern analysis) and protein production (by ELISA) in primary human trabecular osteoblasts (hOB) obtained from healthy donors. Genistein increased OPG mRNA levels and protein secretion by hOB cells by up to two‐ to six‐fold in a dose‐ (P < 0.0001) and time‐dependent (P < 0.0001) fashion with a maximum effect at 10−7 M. Co‐treatment with the pure ER antagonist ICI 182,780 completely abrogated the stimulatory effects of genistein on OPG protein secretion, indicating that these effects were specific and directly mediated through the ER. Pre‐treatment with genistein partially prevented the inhibitory effects of the glucocorticoid dexamethasone on OPG mRNA and protein production. The stimulation of OPG mRNA levels by genistein was not affected by the protein synthesis inhibitor, cycloheximide and was shown to be due to enhancement of OPG gene transcription. In conclusion, our data suggest that the phytoestrogen genistein is capable of upregulating the production of OPG by human osteoblasts. Thus, dietary sources of phytoestrogens may help to prevent bone resorption and bone loss by enhanced osteoblastic production of OPG. J. Cell. Biochem. 84: 725–735, 2002.

Tape functionality: sonographic tape characteristics and outcome after TVT incontinence surgery.

To investigate tension‐free vaginal tape (TVT) position and shape using ultrasound (US) and correlate the findings to outcome.

Atorvastatin stimulates the production of osteoprotegerin by human osteoblasts

Recently, HMG‐CoA reductase inhibitors (statins), potent inhibitors of cholesterol biosynthesis, have been linked to protective effects on bone metabolism. Because of their widespread use, prevention of bone loss and fractures would be a desirable side effect. However, the mechanisms how statins may affect bone metabolism are poorly defined. Here, we evaluated the effect of atorvastatin on osteoblastic production of receptor activator of nuclear factor‐κB ligand (RANKL) and osteoprotegerin (OPG), cytokines that are essential for osteoclast cell biology. While RANKL enhances osteoclast formation and activation, thereby, promoting bone loss, OPG acts as a soluble decoy receptor and antagonizes the effects of RANKL. In primary human osteoblasts (hOB), atorvastatin increased OPG mRNA levels and protein secretion by hOB by up to three fold in a dose‐dependent manner with a maximum effect at 10−6 M (P < 0.001). Time course experiments indicated a time‐dependent stimulatory effect of atorvastatin on OPG mRNA levels after 24 h and on OPG protein secretion after 48–72 h (P < 0.001). Treatment of hOB with substrates of cholesterol biosynthesis that are downstream of the HMG‐CoA reductase reaction (mevalonate, geranylgeranyl pyrophosphate) reversed atorvastatin‐induced enhancement of OPG production. Of note, atorvastatin abrogated the inhibitory effect of glucocorticoids on OPG production. Treatment of hOB with atorvastatin enhanced the expression of osteoblastic differentiation markers, alkaline phosphatase and osteocalcin. In summary, our data suggest that atorvastatin enhances osteoblastic differentiation and production of OPG. This may contribute to the bone‐sparing effects of statins.

Effects of oral contraceptives on circulating osteoprotegerin and soluble RANK ligand serum levels in healthy young women

objective  Osteoprotegerin (OPG) represents a secreted cytokine which regulates bone mass by blocking receptor activator of nuclear factor‐κB ligand (RANKL), the principal regulator of osteoclast function. In vitro, OPG production is upregulated by oestrogens in osteoblastic lineage cells, a mechanism that has been discussed as a protective paracrine mechanism of oestrogens on the skeleton. To define the effects of oestrogens on the RANKL/OPG system in vivo, we evaluated OPG and both free and total soluble RANKL (sRANKL) serum levels in healthy young women with or without oral contraceptives.

Cytokines, Osteoprotegerin, and RANKL In Vitro and Histomorphometric Indices of Bone Turnover in Patients With Different Bone Diseases†

Cytokines are supposed to play an essential role in the regulation of the bone metabolic unit. However, information on cytokine production of primary human osteoblasts from patients with metabolic bone disease is scarce, and few attempts have been made to correlate such data to histomorphometric parameters of individual patients. We investigated 11 patients with metabolic bone disease referred to our outpatient department for bone biopsy and analyzed interleukin (IL)‐1, IL‐6, and TNF‐α protein release and gene expression in primary osteoblast cultures. Compared with four controls, five patients showed normal cytokine protein release, whereas six patients showed much higher levels of interleukin‐6 (26‐fold) and TNF‐α (84‐fold). All three cytokines were strongly correlated concerning gene expression and/or protein levels (r = 0.72–0.96). Histomorphometric analysis of the bone samples showed that eroded surface (ES/BS) as a parameter of bone resorption was significantly associated with TNF‐α. In addition, RANKL gene expression was positively associated with ES/BS and osteoclast surface (Oc.S/BS). Finally, the formation parameters osteoid volume and osteoid surface were negatively associated with TNF‐α. In conclusion, in an in vitro‐ex vivo model of bone cells obtained from a group of 11 patients with different forms of metabolic bone disease, cytokine release in conditioned medium was significantly associated with bone resorption and bone formation, as quantified by histomorphometry. TNF‐α seemed to be the more important cytokine; its effect on bone resorption could be mediated by RANKL.

Role of bladder neck mobility and urethral closure pressure in predicting outcome of tension-free vaginal tape (TVT) procedure

To investigate how urethral mobility and urethral closure pressure affect the outcome of tension‐free vaginal tape (TVT) insertion for stress incontinence.

Black cohosh: just another phytoestrogen?

Most clinical trials on phytoestrogens detected no significant effects on classic menopausal symptoms. Soy-rich diets positively influence certain aspects (e.g. sleep disturbances and mood swings), but the level of relief is low. Duration of the specific diet before menopause onset predicts the extent of the benefit. Isoflavones exert competitive effects at the estrogen receptor and might interfere with anti-proliferative properties of endocrine anti-cancer therapies. Clinical trials on effects of Actaea racemosa (black cohosh) extracts on menopausal symptoms have yielded more positive results. Two recent studies showed excellent efficacy against classic menopausal complaints and osteoprotective properties, and extracts were deemed safe even when the dosage was increased threefold. Furthermore, several studies suggest that A. racemosa extracts might help control psychic problems typically found during menopausal transition.

Alkaline phosphatase expression during monocyte differentiation. Overlapping markers as a link between monocytic cells, dendritic cells, osteoclasts and osteoblasts.

Human monocytes (Mo) in culture can be differentiated into macrophages (M phi), dendritic cells (DC) and osteoclasts. In addition, we have established a Mo-derived in vitro granuloma model which here was compared with ex-vivo isolated foreign body granuloma cells. In these models overlapping phenotypes developed between monocyte-derived dendritic cells (MoDC), osteoclasts, M phi, and osteoblasts. In Mo cultures granulomas were induced by immobilized particulate material. AP activity (osteoblast marker) was found to be co-expressed with cytoplasmic tartrate resistant acid phosphatase (TRAP) as a marker of osteoclasts. While proliferating, the number of AP+ cells decreased, being replaced by cells co-expressing the osteoclast markers vitronectin receptor (VNR) and TRAP. Coexpression of the Mo/M phi marker CD68 with AP or VNR confirmed the monocytic origin of the cells. When Mo were treated with interleukin-4 (IL-4), the number of AP+ cells markedly increased and remained stably expressed over 12 days. In explants from ex vivo granulomas obtained from endoprosthetic revisions the major cell type was the AP+ cell co-expressing CD68. The bone-specific alkaline phosphatase (BAP) as a marker of osteoblasts was detected by FACS analysis in the ex vivo granuloma cells. By RT-PCR the mRNA for osteocalcin, which is a highly specific marker for osteoblasts, was detected. From our results we conclude an ontogenetic relationship between macrophages, DC and osteoclasts. Furthermore, the data suggest a transdifferentiation between Mo and osteoblasts.