Expression of the vitamin D receptor, of estrogen and thyroid hormone receptor alpha- and beta-isoforms, and of the androgen receptor in cultures of native mouse bone marrow and of stromal/osteoblastic cells.
ABSTRACT Marrow stromal cells mediate the effect of 1alpha,25-dihydroxyvitamin D3 on formation of osteoclast-like cells from undifferentiated hematopoetic precursors in bone marrow. Induction by the vitamin D hormone of multinucleated, calcitonin receptor- and tartrate-resistant acid phosphatase-positive cells in primary mouse bone marrow culture can be modulated by other members of the steroid/thyroid hormone family, such as triiodothyronine, which has a positive effect, as well as 17beta-estradiol and 5alpha-dihydrotestosterone, which both act as inhibitors of osteoclastogenesis. In an attempt to relate these effects of the steroid/thyroid hormones to the presence of their respective nuclear receptors, we studied expression of the vitamin D receptor (VDR), estrogen receptor (ER)-alpha and -beta, thyroid hormone receptor (TR)-alpha and -beta, and androgen receptor (AR) in total bone marrow as well as primary marrow stromal cell cultures. By using reverse-transcriptase-polymerase chain reaction, in both cases amplification products were obtained, which were identified by multiple restriction fragment length analysis as transcripts from mRNA specific for the ligand-binding domains of the VDR, ER-alpha, ER-beta, TR-alpha, TR-beta, and AR. Specific immunostaining by indirect peroxidase labeling revealed that among the various cell types present in bone marrow, the steroid/ thyroid hormone receptors are abundant particularly in marrow stromal cells. In another series of experiments, we extended our survey on receptor expression also to stromal/osteoblastic cell lines. At the mRNA level, the complete repertoire of steroid/thyroid hormone receptors was present in preadipocytic ST2 cells as well as in osteoblastic MC3T3-E1 cells. By immunocytochemical staining of the latter, it became apparent that single cells exhibit wide variations in intensity of specific signals for all the receptors investigated, so that, notably in contrast to primary stromal cells and ST2 cells, MC3T3-E1 display a mosaic pattern of receptor protein expression.
Article: The effects of Ankaferd (R) Blood Stopper on transcription factors in HUVEC and the erythrocyte protein profile[show abstract] [hide abstract]
ABSTRACT: Objective: Ankaferd (R) Blood Stopper (ABS) is an herbal extract that has historically been used as a hemostatic agent in traditional Turkish medicine. ABS is comprised of a standardized herbal mixture of T vulgaris, G. glabra, V vinifera, A. officinarum, and U. dioica. ABS's basic mechanism of action is the formation of an encapsulated protein web, which represents the focal point for vital erythrocyte masses. The hemostatic effects of ABS have been observed in vitro and in vivo. ABS was registered as a hemostatic agent for external hemorrhages and dental bleeding following phase I randomized, double-blind crossover placebo-controlled clinical research, and safety and efficacy reports. In terms of the potential use of ABS, transcription factors may be novel factors that play a role in the hemostatic and other pleiotropic effects of ABS. Materials and Methods: Hence, the present study aimed to investigate the effects of ABS on endothelium, and possible transcription factor changes in HUVEC (human umbilical vein endothelial cells) and the erythrocyte membrane profile. ABS (5 mu L and 50 mu L) was administered to HUVEC (in 75 cm(2); similar to 75% fullness) for 5 min and 15 min. Results: ABS caused significant increases in the level of activation of the following transcription factors; AP2, AR, CRE/ATF1, CREB, E2F1-5, E2F6, EGR, GATA, HNF-1, ISRE, Myc-Max, NF-1, NFkB, p53, PPAR, SMAD 2/3, SP1, TRE/AP1, and YY1. Following erythrocyte membrane isolation, protein complexes were undissolved, but denatured. The protein complex formed was resistant to heat and detergent. Trypsin and sonication were used in order to break this complex; the complex dissolved and erythrocyte membrane proteins were released in SDS-PAGE. Conclusion: ABS established a very fast and solid protein web, and increased the level of transcription factor activation. Therefore the cellular effects of ABS could be related to different intracellular biological pathways. (Turk J Hematol 2011; 28: 276-85)Turkish Journal of Hematology. 01/2011; 28(4):276-285.
Article: Altered TNSALP expression and phosphate regulation contribute to reduced mineralization in mice lacking androgen receptor.[show abstract] [hide abstract]
ABSTRACT: While androgen receptor (AR)-deficient mice developed osteopenia in endochondral bones due to the high bone turnover with increased bone resorption by osteoclasts, little is known about the mechanism of intramembranous bone loss contributed by AR in osteoblasts. Here, we discovered a dramatic decrease in the area of calcification, new bone, and the number of osteocytes in calvaria from AR-deficient mice related to a reduction in mineralization caused, in part, by the diminished activity of AR-deficient osteoblasts. Enforced AR expression in differentiated osteoblasts boosts mineralization while knockdown of AR expression prevents androgen-induced mineralization. We identified the tissue-nonspecific alkaline phosphatase (TNSALP) and several members of small integrin binding ligand N-linked glycoprotein (SIBLING) gene family as androgen target genes required for AR-mediated bone formation. We show that inorganic phosphate (P(i)) levels and TNSALP activity increased in response to androgen/AR and P(i) signals increase the expression and translocation of AR. The ectopic expression of TNSALP or P(i) partially rescued the bone loss due to AR deficiency. Thus, androgen/AR signaling plays an essential role in bone formation by coordinating the expression of genes associated with phosphate regulation.Molecular and cellular biology 11/2008; 28(24):7354-67. · 6.06 Impact Factor
Article: The thyroid hormone receptor (TR) beta-selective agonist GC-1 inhibits proliferation but induces differentiation and TR beta mRNA expression in mouse and rat osteoblast-like cells.[show abstract] [hide abstract]
ABSTRACT: Previous studies showed anabolic effects of GC-1, a triiodothyronine (T3) analogue that is selective for both binding and activation functions of thyroid hormone receptor (TR) beta1 over TRalpha1, on bone tissue in vivo. The aim of this study was to investigate the responsiveness of rat (ROS17/2.8) and mouse (MC3T3-E1) osteoblast-like cells to GC-1. As expected, T3 inhibited cellular proliferation and stimulated mRNA expression of osteocalcin or alkaline phosphatase in both cell lineages. Whereas equimolar doses of T3 and GC-1 equally affected these parameters in ROS17/2.8 cells, the effects of GC-1 were more modest compared to those of T3 in MC3T3-E1 cells. Interestingly, we showed that there is higher expression of TRalpha1 than TRbeta1 mRNA in rat (approximately 20-90%) and mouse (approximately 90-98%) cell lineages and that this difference is even higher in mouse cells, which highlights the importance of TRalpha1 to bone physiology and may partially explain the modest effects of GC-1 in comparison with T3 in MC3T3-E1 cells. Nevertheless, we showed that TRbeta1 mRNA expression increases (approximately 2.8- to 4.3-fold) as osteoblastic cells undergo maturation, suggesting a key role of TRbeta1 in mediating T3 effects in the bone forming cells, especially in mature osteoblasts. It is noteworthy that T3 and GC-1 induced TRbeta1 mRNA expression to a similar extent in both cell lineages (approximately 2- to 4-fold), indicating that both ligands may modulate the responsiveness of osteoblasts to T3. Taken together, these data show that TRbeta selective T3 analogues have the potential to directly induce the differentiation and activity of osteoblasts.Calcified Tissue International 05/2009; 84(4):324-33. · 2.38 Impact Factor