In seven strains of cultured normal human osteoblast-like cells, a mean of 1615 molecules of tritium-labeled 17 beta-estradiol per cell nucleus could be bound to specific nuclear sites. The nuclear binding of the labeled steroid was temperature-dependent, steroid-specific, saturable, and cell type-specific. These are characteristics of biologically active estrogen receptors. Pretreatment with 10 nanomolar estradiol in vitro increased the specific nuclear binding of progesterone in four of six cell strains, indicating an induction of functional progesterone receptors. RNA blot analysis demonstrated the presence of messenger RNA for the human estrogen receptor. The data suggest that estrogen acts directly on human bone cells through a classical estrogen receptor-mediated mechanism.

Evidence of estrogen receptors in normal human osteoblast-like cells

Medullary bone forms in egg-laying birds in response to gonadal steroids and is the most overtly oestrogen-dependent of all bone types. It acts as a labile reservoir for the supply of eggshell calcium. Previous studies indicate that feeding calcium- and vitamin-D-deficient diets to chickens results in resorption of cortical rather than medullary bone. More recent studies in calcium-stressed quail hens question this hypothesis and suggest that during the first 2 weeks of dietary calcium depletion the medullary bone is resorbed while cortical bone volume remains intact. The role of the osteoclast in bone resorption is the focus of much research that has recently included studies of medullary bone osteoclasts. The functional morphology of the avian cells, i.e. changes from quiescent to active osteoclasts with ruffled borders, reflects the rapid changes in medullary bone turnover that occur during the egg-laying cycle. Unlike mammalian osteoclasts, those from avian sources generally appear refractory to inhibitory factors such as calcitonin or raised extracellular calcium concentration. However, medullary bone osteoclasts cultured in vitro for several days recover their ability to respond to the latter factor by increasing their levels of free cytosolic Ca2+, reducing tartrate-resistant acid phosphatase secretion and reducing their cell spread area. It is suggested that factors such as ambient calcium levels and prostaglandins may form part of a system of rapid local control for medullary bone osteoclast activity.

Medullary bone and avian calcium regulation

Estrogen is essential for the development and maintenance of optimal bone mass in women and men, and acts through activation of estrogen receptors (ER). We have examined the pathways of estrogen action on the skeleton by seeking to localize the "classical" estrogen receptor, ER alpha, to particular cells to test the hypotheses that 1) estrogen directly influences growth plate chondrocytes; and 2) estrogen has a principal action on bone tissue via osteoblasts. ER alpha messenger ribonucleic acid (mRNA) was localized by in situ hybridization in human specimens from five males (11-15 yr old), two females (9 and 11 yr old), and three growing rabbits. In all of the human material examined, ER alpha mRNA was consistently identified in chondrocytes. In all of the rabbit tissue studied, ER alpha mRNA was localized in chondrocytes of the growth plate and the subarticular epiphyseal growth center. ER alpha mRNA signals were readily observed in both active osteoblasts and lining cells on trabecular surfaces of all samples. No clear evidence of positive staining was detectable in osteoclasts or osteocytes in either species. The distribution of ER alpha mRNA coincided with immunolocalization of the ER protein in the human specimens. These data suggest a direct action of estrogen on growth plate chondrocytes that may affect longitudinal growth and subsequent fusion of the growth plate and also on osteoblasts to affect bone formation at trabecular sites.

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Localization of estrogen receptor-alpha in human and rabbit skeletal tissues.

Although estrogens profoundly influence skeletal growth and maturation, their mechanism of action is still unclear To identify their target cells in bone, estrogen receptors were located by immunofluorescence using the H222 monoclonal antibody in cryosections (both undecalcified and briefly decalcified) of hyperplastic mandibular condyle (persistent asymmetric mandibular growth) from a 14-year-old girl and radius and ulna from an 18-month-old female pig (epiphyseal fusion) and from a 3-month-old guinea pig (epiphyses open) Bone was removed from the animals at the peak of estrus The most striking feature in all three species was the high proportion (approximately 50%) of receptor positive osteocytes Although all sections contained active bone-forming surfaces, we were unable to identify clearly osteoblasts or lining cells that were estrogen receptor positive In pig bone only, distinctive groups of receptor positive chondrocytes, with a pericellular localization of collagen type 1, were detected above the growth plate but below secondary centers of ossification This observation suggests that osteocytes are major skeletal estrogen target cells and may be involved in coordinating the response of surface bone cells to the hormone, and further that chondrocytes may be involved in estrogen-induced epiphyseal growth plate fusion

Preliminary in situ identification of estrogen target cells in bone.

Osteocytes are the most abundant and longest-living cells in the adult skeleton For a long time, osteocytes were considered static and inactive cells, but in recent years, it has been suggested that they represent the key responder to various stimuli that regulate bone formation and remodelling as well as one of the key endocrine regulators of bone metabolism Osteocytes respond to mechanical stimuli by producing and secreting several signalling molecules, such as nitric oxide and prostaglandin E(2) , that initiate local bone remodelling Moreover, they can control bone formation by modulating the WNT signalling pathway, an essential regulator of cell fate and commitment, as they represent the main source of sclerostin, a negative regulator of bone formation Osteocytes can also act as an endocrine organ by releasing fibroblast growth factor 23 and several other proteins (DMP-1, MEPE, PHEX) that regulate phosphate metabolism It has been demonstrated that various bone diseases are associated with osteocyte abnormalities, although it is not clear if these changes are the direct cause of the pathology or if they are secondary to the pathological changes in the bone microenvironment Thus, a better understanding of these cells could offer exciting opportunities for new advances in the prevention and management of different bone diseases

Osteocytes: central conductors of bone biology in normal and pathological conditions.

The localization of estrogen receptors (ERs) in osteogenic cells was immunoelectron microscopically examined in the femurs of female and estrogen-treated male Japanese quail. An electron dense reaction product showing ER localization was observed in the nuclei of osteoblasts and immature osteocytes in the medullary bone of the female quail. However, reaction product was not seen in the osteoclasts. On the endosteal bone surface of male quail, nuclear reaction product was detected in bone lining cells. After 24 h of estrogen treatment, reaction product was observed in the nuclei of preosteoblasts on the endosteal bone surface. After 48 h, the medullary bone partly appeared along the endosteal surface. Nuclear reaction product was seen in osteoblasts on the medullary bone surface.

S Kusuhara

Papers

Immunoelectron microscopic demonstration of estrogen receptors in osteogenic cells of Japanese quail.