Fitzpatrick, Lorraine A.

Abstract The microanatomic distribution of several noncollagenous proteins (NCPs) in bone matrix was examined by immunohistochemical analysis of glycol-methyl methacrylate-embedded normal adult human bone biopsies. Osteopontin and bone sialoprotein stained throughout the lamellae of both trabecular and cortical bone. Cement lines (cortical and trabecular) and the mineralized matrix immediately adjacent to each Haversian canal were intensely stained. Osteocalcin was detected in cement lines; however, lamellar staining varied depending on the location within the individual unit of bone. In cortical bone, the inner concentric lamellae of osteons were often unstained but the outer lamellae were heavily stained for osteocalcin. Osteonectin was not detected in cement lines and in most specimens revealed a pattern similar to that of osteocalcin with respect to the absence of immunostaining within the inner concentric lamellae. Decorin was prominent in the perilacunar matrix, the canaliculi of osteocytes, and the matrix immediately adjacent to quiescent Haversian canals. Biglycan appeared evenly distributed throughout cortical and trabecular bone matrix. These results suggest that the incorporation of NCPs into matrix may vary depending on the stage of formation of individual bone units. The specific distribution and spatial relationship of these NCPs may be related to the function of each protein during bone resorption and formation. The distinct patterns of NCP localization in bone support the hypothesis that in addition to their structural and mineral-inducing properties, these proteins may influence the events associated with bone remodeling, such as recruitment, attachment, differentiation, and activity of bone cells.

Abstract Cell-cell interactions are important in the regulation of endocrine cell secretion. To investigate the possibility that cell communication may alter the regulation of parathyroid cell secretion, we utilized the reverse hemolytic plaque assay (RHPA) to measure parathyroid hormone (PTH) release from individual cells. Bovine parathyroid cells were dispersed and plated with protein A-conjugated erythrocytes at cell densities ranging from 0.9 to 36 X 102 cells/cm2 in 0.2 mM calcium. Cell populations were greater than 98% homogeneous as determined by immunocytochemistry and in situ hybridization for PTH mRNA. Plaques were developed and data analyzed for the amount of PTH per cell released (plaque area in μm2 X 104) and the determination of cell recruitment (% plaques formed). A positive correlation existed between parathyroid cell density and the amount of PTH released. As the distance between cells increased, the plaque area (amount of PTH released per cell) decreased (ranging from 1.0 X 1044 μm2 at 0.9 X 102 cells/cm2 versus 1.6 X 104 μm2 at 36 X 102 cells/cm2). The percentage of cells releasing PTH (recruitment) also decreased (16% at 0.9 X 102 cells/cm2 versus 47% at 36 X 102 cells/cm2). These data suggest that parathyroid cells in close proximity are stimulated to secrete more hormone than those at lesser densities. In addition, parathyroid cells are recruited to secrete PTH when plated at high density. Factor(s) released by the parathyroid cell may increase cell responsiveness and stimulate secretion in a paracrine fashion.

Introduction The earliest lesion in the kidneys of idiopathic calcium oxalate stone formers is deposition of calcium phosphate in the interstitium, termed a Randall's plaque. Yet the cellular and molecular factors leading to their formation are unknown. Methods The influence of urinary proteins on adhesion of preformed calcium oxalate crystals to rat continuous inner medullary collecting duct (cIMCD) cells was studied in vitro, and cIMCD cells were also exposed to calcifying media containing β-glycerophosphate for up to 28 days. Renal tissue was obtained from a stone-forming and non-stone-forming individual at the time of nephrectomy. These nanoparticles, isolated from renal stones obtained at the time of surgical resection, were analyzed and propagated in standard cell culture medium. Results Urinary proteins influence crystal adhesion to renal epithelial cells, and this activity is abnormal in the urine of stone-forming patients. cIMCD cells assumed an osteoblastic phenotype when exposed to the calcifying medium, expressing two bone matrix proteins (osteopontin and bone sialoprotein) that were also identified in the kidney of the stone-forming patient and associated with crystal deposition. Nanoparticles were propagated from the majority of renal stones. Isolates were susceptible to selected metabolic inhibitors and antibiotics and contained conserved bacterial proteins and deoxyribonucleic acid (DNA). Conclusions These results suggest new paradigms for Randall's plaque formation and idiopathic calcium oxalate stone disease. It seems unlikely that these events are driven solely by physical chemistry; rather, they are critically influenced by specific proteins and cellular responses, and understanding these events will provide clues toward novel therapeutic targets.

Premenopausal women have a significant reduction in coronary artery disease compared to age matched males. Little is known about the mechanism underlying this cardio protective effect of estrogen. Contradictory evidence has been published and our lack of basic understanding of hormone interactions and bioavailability of different estrogens prevents definitive interpretation of these data. We demonstrate gender-specific effects in the proliferation of coronary artery vascular smooth muscle cells obtained from a sexually mature animal model. Vascular smooth muscle cells are an integral component of the atherosclerotic plaque, and inhibition of cell proliferation by estrogen may be one mechanism by which estrogen exerts its cardio protective effect. Various types of estrogen may also have different mechanistic actions on the vascular system. No differences are demonstrated in overall estradiol binding in vascular smooth muscle cells obtained from male or female animals; however, differences in c-jun, c-fos and TIEG gene expression were gender related. Inhibition of vascular smooth muscle cell proliferation may have important implications in the prevention of atherosclerotic disease and these studies may provide evidence for the cardio protective effect of estrogen.

The decrease in estrogen levels that follows the onset of menopause results in rapid bone loss and osteoporosis. The major effect of estrogen deficiency on bone metabolism is an increase in the rate of bone resorption, but the precise mechanism by which this occurs remains unresolved. A recently developed technique for the isolation of avian osteoclasts has been modified to obtain highly purified multinucleated cells from human giant cell tumors. These osteoclast-like cells have been examined for evidence of estrogen receptors (ERs) and responses to 17 beta-estradiol (17 beta-E2). Analysis of giant-cell RNA demonstrated expression of ER mRNA. Furthermore, immunoblot analysis revealed that the giant cells contained a 66-kDa protein that was recognized by a monoclonal antibody specific for the human ER. When isolated multinucleated cells were cultured on slices of bone, there was a dose-dependent decrease in resorption in response to treatment detectable at 10 pM 17 beta-E2. Treatment with 10 nM 17 alpha-estradiol or vehicle (control) did not inhibit resorption. Moreover, the multinucleated cells isolated from these tumors had decreased mRNA levels for cathepsin B, cathepsin D, and tartrate-resistant acid phosphatase (TRAP) as well as secreted cathepsin B and TRAP enzyme activity in response to treatment with 10 nM 17 beta-E2. In contrast to these data, no change in gene expression was detected in mononuclear cells from these tumors in response to 17 beta-E2 treatment. These data support the proposition that human osteoclasts are target cells for estrogen and that estrogen can inhibit bone resorption by human osteoclasts.

Abstract Isolated osteoclasts from 5-week-old chickens respond to estradiol treatment in vitro with decreased resorption activity, increased nuclear proto-oncogene expression, and decreased lysosomal enzyme secretion. This study examines osteoclasts from embryonic chickens and egg-laying hens for evidence of estrogen responsiveness. Although osteoclasts from both of these sources express estrogen receptor mRNA and protein, estradiol treatment had no effect on resorption activity. In contrast to the lack of effect on resorption, estradiol treatment for 30 minutes resulted in steady-state mRNA levels of c-fos and c-jun increasing in osteoclasts from embryonic chickens and decreasing in osteoclasts from egg-laying hens. These data suggest that a nuclear proto-oncogene response may not be involved in estradiol-mediated decreased osteoclast resorption activity. To examine the influence of circulating estrogen on osteoclast estrogen responsiveness, 5-week-old chickens were injected with estrogen for 4 days prior to sacrifice. Estradiol treatment of osteoclasts from these chickens did not decrease resorption activity in vitro. Transfection of an estrogen receptor expression vector into osteoclasts from the estradiol-injected chickens and egg-laying hens restored estrogen responsiveness. Osteoclasts from 5-week-old chickens and estradiol treated 5-week-old chickens transfected with the estrogen receptor expression vector contained significantly higher levels of estrogen receptor protein and responded to estradiol treatment by decreasing secretion of cathepsins B and L and tartrate-resistant acid phosphatase. In contrast, osteoclasts from embryonic chickens, egg-laying hens, and estradiol-treated 5-week-old chickens either untransfected or transfected with an empty expression vector did not respond similarly. These data suggest that modulation of osteoclast estrogen responsiveness may be controlled by changes in the osteoclast estrogen receptor levels.

Abstract Background: Controversy exists over the association of estrogen and cardiovascular disease. Estrogen receptors (ERs) α and β are expressed in the endothelial cells and vascular smooth muscle cells (VSMCs) of many arteries, but the relative importance of ERα or ERβ in mediating the vascular response to estrogens is not well defined, particularly in humans. We have shown previously that postmenopausal women receiving hormone therapy (HT) had lower mean coronary artery calcium, plaque area, and calcium-to-plaque ratio compared with untreated women. In this study, we examined coronary artery ERα and ERβ expression in pre- and postmenopausal women as a function of plaque area, calcium area, calcium-to-plaque ratio, and estrogen status. Methods: Coronary arteries were obtained at autopsy from a total of 55 women: nine premenopausal women, 13 postmenopausal women on HT and 33 untreated postmenopausal women (non-HT). Coronary calcification was quantified by contact microradiography, and atherosclerotic plaque area was measured histologically. Coronary artery cross-sections were immunostained for ERα and ERβ, and the amount of receptors was estimated semiquantitatively in each arterial wall layer (intima, adventitia, and media). Double immunofluorescence was used to colocalize ERα and ERβ with smooth muscle actin, a marker of VSMCs. Results: ERβ and ERα were expressed in all artery wall layers, but most avidly in the media (P = 0.001), and colocalized with VSMCs. ERβ expression exceeded ERα expression in all wall layers (P < 0.001) and was adjacent to areas of calcium deposition. ERβ expression in the intimal layer correlated with calcium content, plaque area, and calcium-to-plaque ratio (all P < 0.01) and tended to be greater in non-HT than in HT women (P = 0.06). ERα expression did not vary significantly among groups, nor did it correlate with calcium content, plaque area or calcium-to-plaque ratio. Expression of ERα but not ERβ declined with age (P < 0.01) in HT women only. Age had no effect on ERα or ERβ expression in non-HT or premenopausal women. Conclusions: ERβ is the predominant ER in human coronary arteries and correlates with coronary calcification, a marker of severe atherosclerosis. Increased ERβ expression is linked to advanced atherosclerosis and calcification independent of age or hormone status. Future pharmacogenetic studies that target this receptor are needed to confirm causality.