Biomedical subjects

Jonathan H Tobias

Publications and source records attributed to Jonathan H Tobias.

6 recordsLinked to original sources

PubMed2003

Transcriptional regulation of a BMP-6 promoter by estrogen receptor alpha.

UNLABELLED: The effects of 17beta-estradiol (E2) and ICI 182,780 (ICI) on activity of a BMP-6 promoter were compared in osteoblast-like and breast cancer cells transiently transfected with ERalpha. E2 but not ICI stimulated BMP-6 reporter activity in breast cancer cells, whereas the opposite was observed in osteoblast-like cells, associated with lack of AF-2 dependence of the response, and absent intranuclear localization of ERalpha, suggesting the involvement of a distinct ERalpha-dependent response mechanism in osteoblasts. INTRODUCTION: Previous studies suggest that the tissue-selective effect of antiestrogens on bone reflects the ability of these compounds to target certain osteoblast regulatory genes. To explore this hypothesis, we examined whether antiestrogens preferentially stimulate the bone morphogenetic protein 6 (BMP-6) promoter in bone cells, and if so, whether this activity is associated with a distinct estrogen receptor (ER)alpha-dependent response mechanism to that in other cell types. MATERIALS AND METHODS: We compared the effects of 17beta-estradiol (E2) and ICI 182,780 (ICI) on activity of a 4.3-kb BMP-6 reporter construct in osteoblast-like cells (human MG63 and SaOS-2 cells and rat ROS 17/2.8 cells), human MCF-7 and T47-D breast cancer cell lines, and HepG2 hepatoma cells, after transient transfection with ERalpha, ERbeta, and mutant ER constructs. RESULTS: E2, but not ICI, stimulated BMP-6 reporter activity by approximately 100% in MCF-7, T47-D cells, and HepG2 cells when transfected with ERalpha. In contrast, in ERalpha-transfected osteoblast-like cells, an increase in reporter activity of approximately 75% was observed after treatment with ICI but not E2. The response of MG63 cells to ICI and MCF-7 cells to E2 both required ERalpha as opposed to ERbeta and the ERalpha activation function (AF)-1 activation domain. However, whereas the AF-2 domain was also required for E2 to stimulate reporter activity in MCF-7 cells, the response to ICI in MG63 cells was AF-2 independent. In further studies where we compared the intracellular distribution of ERalpha associated with these responses, E2-dependent stimulation of the BMP-6 reporter in MCF-7 cells was associated with intranuclear localization of ERalpha, whereas extranuclear localization was seen in rat osteosarcoma cells (ROS) cells treated with ICI. CONCLUSIONS: Antiestrogens selectively stimulate BMP-6 reporter activity in osteoblast-like cells through a distinct ERalpha-dependent mechanism characterized by independence of the AF-2 domain and extranuclear localization of ERalpha.

Animals↗

PubMed2003

PTH/PTHrP activity and the programming of skeletal development in utero.

There is increasing evidence that nutritional deficiency in utero adversely affects bone development and the risk of developing osteoporosis in later life. Although the mechanisms involved are unknown, circumstantial evidence points to an important role of PTH/PTHrP activity. It is recognized that PTH and PTHrP are critically involved in regulating fetal calcium homeostasis, actions that are mediated at least in part by PPR. As well as playing a central role in the maintenance of calcium homeostasis in the fetus, studies in transgenic mice show that PTH, PTHrP, and PPR exert similar effects on skeletal development in utero, acting to increase the size of the trabecular envelope and decrease that of the cortical envelopes. Taken together, these observations raise the possibility that stimulation of PTH/PTHrP activity in the fetus in response to calcium deficiency acts to increase the size of the trabecular envelope but to reduce that of the cortical envelope. Although any increase in trabecular bone at birth is likely to be relatively transient, a decrease in size of the cortical envelope may have a persistent effect on the trajectory of bone growth in subsequent childhood. Consistent with this proposal, preliminary findings from birth cohort studies suggest that maternal calcium intake and cord blood calcium levels are positively related to bone mass of the offspring as assessed later in childhood. Further studies are justified to determine whether alterations in fetal PTH/ PTHrP activity caused by calcium stress lead to a reduction in size of the cortical envelope at birth that persists into childhood and later adult life and to identify modifiable maternal factors that are responsible for these changes.

Bone Development↗

PubMed2003

At the crossroads of skeletal responses to estrogen and exercise.

The recent observation that mice deficient in estrogen receptor alpha (ERalpha) have an impaired response to mechanical strain suggests that ERalpha plays an important role in mediating the response of the skeleton to mechanical loading as well as to estrogen. In view of previous findings that estrogen deficiency leads to a fall in ERalpha numbers, postmenopausal bone loss might result from the impaired response of bone to mechanical strain caused by deficient ERalpha signalling.

Animals↗

PubMed2003

Estrogen receptor-alpha dependency of estrogen's stimulatory action on cancellous bone formation in male mice.

We examined whether estrogen receptor (ER)alpha is required for estrogen to stimulate cancellous bone formation in long bones of male mice. 17 beta-Estradiol (E(2)) was administered to ER alpha(-/-) male mice or wild-type (WT) littermate controls at 40, 400, or 4000 microg/kg by daily sc injection for 28 d and histomorphometric analysis performed at the distal femoral metaphysis. In WT mice, treatment with E(2) (40 microg/kg per d) increased the proportion of cancellous bone surfaces undergoing mineralization and stimulated mineral apposition rate. In addition, higher doses of E(2) induced the formation of new cancellous bone formation surfaces in WT mice. In contrast, E(2) had little effect on any of these parameters in ER alpha(-/-) mice. Immunohistochemistry was subsequently performed using an ER alpha-specific C-terminal polyclonal antibody. In WT mice, ER alpha was expressed both by cancellous osteoblasts and a significant proportion of mononuclear bone marrow cells. Immunoreactivity was also observed in cancellous osteoblasts of ER alpha(-/-) mice, resulting from expression of the activation function-1-deficient 46-kDa ER alpha isoform previously reported to be expressed in normal osteoblasts and bones of ER alpha(-/-) mice. Taken together, our results suggest that estrogen stimulates bone formation in mouse long bones via a mechanism that requires the presence of full-length ER alpha possessing activation function-1.

Animals↗

PubMed2002

Estrogen-induced osteogenesis in mice is associated with the appearance of Cbfa1-expressing bone marrow cells.

Cbfa1 is a transcription factor recognised as being involved in early osteoblast differentiation during embryonic skeletogenesis. To determine whether Cbfa1 plays a similar role in bone formation in the adult, we analysed whether its expression is altered during estrogen-induced osteogenesis, following our recent studies which suggest that this response involves the generation of early osteoblast precursors within bone marrow. To facilitate identification of Cbfa1-expressing cells, these studies were performed in mice heterozygous for a cbfa1 gene deletion (cbfa1(+/-)) using beta-galactosidase (lacZ) as a genetic marker. Cbfa1-expressing cells were identified by lacZ staining of longitudinal sections of the proximal tibial metaphysis. Treatment of cbfa1(+/-) mice with 17beta-estradiol 0.5 mg/week for 24 days led to the appearance of new cancellous bone surfaces. This response was associated with a marked increase in number of Cbfa1-expressing cells within the metaphysis, consisting not only of osteoblasts on bone surfaces but also of cells within the adjacent bone marrow. We subsequently enumerated Cbfa1-expressing cells at earlier time-points following estrogen, in sections co-stained for ALP activity. After 4 days of estrogen treatment, a population of cells appeared within the marrow cavity which expressed Cbfa1, but were negative for ALP. At later time-points, large numbers of Cbfa1 + bone marrow cells were still present, but the majority of these were close to new trabecular bone surfaces at sites which showed high levels of ALP activity. An equivalent distribution of Cbfa1-expressing cells was observed in further studies where Cbfa1 expression was analysed in wild-type mice by immunohistochemistry. We conclude that estrogen-induced osteogenesis is associated with the appearance of a population of Cbfa1-expressing cells within bone marrow, which we hypothesize to represent the osteoblast precursor population responsible for subsequent new bone formation.

Animals↗

PubMed2002

Increased bone morphogenetic protein-6 expression in mouse long bones after estrogen administration.

High-dose estrogen administration is known to induce new bone formation in mouse long bones. To study the role of regulatory proteins in this response, we examined associated changes in femoral messenger RNA (mRNA) for candidate factors. 17beta-estradiol (E2) 0.5 mg was administered to intact female mice by weekly injection, and Northern blot analysis was performed 1, 2, 4, 8, 12, and 16 days after the first injection. In contrast to other factors, an increase was observed in mRNA for bone morphogenetic protein-6 (BMP-6), which reached significance at day 8 and subsequent time-points. Estrogen-induced changes in BMP-6 protein expression were assessed by immunocytochemistry in longitudinal femoral sections. In untreated animals, BMP-6 was expressed by a significant proportion of growth plate chondrocytes and a subpopulation of bone marrow cells. In contrast, osteoblasts were consistently BMP-6 negative. From as early as 4 days after starting estrogen, clusters of slightly elongated BMP-6-positive cells were observed within the marrow cavity; the majority were close to active bone formation surfaces. Double immunolabeling studies revealed that only approximately 10% of BMP-6-positive bone marrow cells co-expressed the osteoblast transcription factor Cbfa1 suggesting that they are largely distinct from the osteoblast precursor population generated concurrently. BMP-6-positive cells expressed neither leukocyte nor erythroid markers (CD45 and TER-119, respectively), consistent with a stromal origin. We conclude that estrogen-induced osteogenesis in female mice is associated with increased levels of BMP-6 mRNA in mouse femurs, which seems to reflect the emergence of clusters of BMP-6 positive stromal cells adjacent to active bone formation surfaces. These findings raise the possibility that BMP-6 serves as a paracrine mediator of estrogen's osteogenic action in mice.

Animals↗