Characterization of circulating osteoblast lineage cells in humans.

We recently identified circulating osteoblastic cells using antibodies to osteocalcin (OCN) or alkaline phosphatase (AP). We now provide a more detailed characterization of these cells. Specifically, we demonstrate that 46% of OCN positive (OCN(pos)) cells express AP, and 37% also express the hematopoietic/endothelial marker CD34. Using two different anti-OCN antibodies and forward/side light scatter characteristics by flow cytometry, we find that OCN(pos) cells consist of two distinct populations: one population exhibits low forward/side scatter, consistent with a small cell phenotype with low granularity, and a second population has higher forward/side scatter (larger and more granular cell). The smaller, low granularity population also co-expresses CD34, whereas the larger, more granular cells are CD34 negative. Using samples from 26 male subjects aged 28 to 68 years, we demonstrate that the concentration of circulating OCN(pos) cells increases as a function of age (R=0.59, P=0.002). By contrast, CD34(pos) cells tend to decrease with age (R=-0.31, P=0.18); as a consequence, the ratio of OCN(pos):CD34(pos) cells also increase significantly with age (R=0.54, P=0.022). These findings suggest significant overlap between circulating cells expressing OCN and those expressing the hematopoietic/endothelial marker CD34. Further studies are needed to define the precise role of circulating OCN(pos) cells not only in bone remodeling but rather also potentially in the response to vascular injury.

Skeletal effects of estrogen are mediated by opposing actions of classical and nonclassical estrogen receptor pathways.

UNLABELLED: ER alpha acts either through classical (ERE-mediated) or nonclassical (non-ERE) pathways. The generation of mice carrying a mutation that eliminates classical ER alpha signaling presents a unique opportunity to study the relative roles of these pathways in bone. This study defines the skeletal phenotype and responses to ovariectomy and estrogen replacement in these mice. INTRODUCTION: Estrogen receptor alpha (ER alpha) can act either through classical estrogen response elements (EREs) or through non-ERE (nonclassical) pathways. To unravel these in bone, we crossed mice heterozygous for a knock-in mutation abolishing ERE binding (nonclassical ER alpha knock-in [NERKI]) with heterozygote ER alpha knockout mice and studied the resulting female ER alpha(+/+), ER alpha(+/NERKI), and ER alpha(-/NERKI) mice. The only ER alpha present in ER alpha(-/NERKI) mice is incapable of activating EREs but can signal through nonclassical pathways, whereas ER alpha(+/NERKI) mice may have a less drastic alteration in the balance between classical and nonclassical estrogen signaling pathways. MATERIALS AND METHODS: BMD was measured using DXA and pQCT at 3 months of age (n = 46-48/genotype). The mice were randomly assigned to sham surgery, ovariectomy, ovariectomy + estradiol (0.25 microg/day), or ovariectomy + estradiol (1.0 microg/day; n = 10-12/group) and restudied 60 days later. RESULTS AND CONCLUSIONS: At 3 months of age, both the ER alpha(+/NERKI) and ER alpha(-/NERKI) mice had deficits in cortical, but not in trabecular, bone. Remarkably, changes in cortical bone after ovariectomy and estrogen replacement in ER alpha(-/NERKI) mice were the opposite of those in ER alpha(+/+) mice. Relative to sham mice, ovariectomized ER alpha(-/NERKI) mice gained more bone (not less, as in ER alpha(+/+) mice), and estrogen suppressed this increase (whereas augmenting it in ER alpha(+/+) mice). Estrogen also had opposite effects on bone formation and resorption parameters on endocortical surfaces in ER alpha(-/NERKI) versus ER alpha(+/+) mice. Collectively, these data show that alteration of the balance between classical and nonclassical ER alpha signaling pathways leads to deficits in cortical bone and also represent the first demonstration, in any tissue, that complete loss of classical ERE signaling can lead to paradoxical responses to estrogen. Our findings strongly support the hypothesis that there exists a balance between classical and nonclassical ER alpha signaling pathways, which, when altered, can result in a markedly aberrant response to estrogen.

Dose-response of estrogen on bone versus the uterus in ovariectomized mice.

BACKGROUND: Estrogen is known to have important effects on both reproductive and non-reproductive tissues. Moreover, there is increasing interest in developing compounds that may have selective effects on bone versus reproductive tissues. METHODS: Since mouse models are often used in these studies, we administrated increasing doses of estradiol (E2) (0 to 500 microg/kg/day) by slow release pellets to ovariectomized 6-month-old C57BL/6 mice and assessed skeletal and uterine responses following 2 months of treatment. RESULTS: The mice lost bone at multiple sites following ovariectomy (OVX); however, while the lowest E2 dose of 5 microg/kg/day completely prevented loss of cancellous bone (at the lumbar spine and tibial metaphysis), it had no stimulatory effects on the uterus. Higher doses of E2 resulted in further increases in bone mineral density, with eventual stimulation of the uterus at a dose of 40 microg/kg/day. By contrast, when 3-month-old C57BL/6 mice were administered the same doses of E2 and studied after 1 month, the 5 microg/kg/day dose resulted in uterine hypertropy, but was not able to prevent loss of cancellous bone. CONCLUSIONS: Thus these results (i) provide data on the dose-response for the effects of E2 on mouse bone and (ii) indicate that the relative effects of E2 on bone versus the uterus are highly dependent on the particular experimental conditions used. This issue needs to be considered in evaluating agents with potential 'selective' effects on bone versus reproductive tissues.

Effects of loss of steroid receptor coactivator-1 on the skeletal response to estrogen in mice.

Steroid receptor coactivator (SRC)-1 is an important nuclear receptor coactivator that enhances estrogen (E) action in many tissues, but its role in mediating E effects on bone is unknown. Thus, we assessed the skeletal response to ovariectomy (ovx) and E replacement in SRC-1 knockout (KO) mice compared with wild-type (WT) littermates. Bone mineral density was measured by dual-energy x-ray absorptiometry and peripheral quantitative computed tomography at baseline and after 2 months of sham surgery, ovx, or ovx plus E replacement. Microcomputed tomography and bone histomorphometry were also performed at the end of the study. Both WT and SRC-1 KO mice lost bone at multiple sites after ovx; however, although an estradiol (E(2)) dose of 10 microg/kg.d completely prevented loss of cancellous bone (at the lumbar spine and tibial metaphysis) in the WT mice, it was entirely ineffective in preventing cancellous bone loss at these sites in the SRC-1 KO mice. This E(2) dose was, however, equally effective on cortical bone in the tibia in the SRC-1 KO and WT mice. Moreover, a 4-fold higher dose of E(2) was able to overcome the deficit in E action in cancellous bone in the SRC-1 KO mice. These findings establish that, in mice, loss of SRC-1 leads to skeletal resistance to E predominantly in cancellous bone.