S. Bord

The effects of estrogen on osteoprotegerin, RANKL, and estrogen receptor expression in human osteoblasts

Estrogen is essential for bone growth and development and for the maintenance of bone health in adulthood. The cellular responses of osteoblasts and osteoclasts to estrogen are initiated via two high-affinity receptors (ERs). Osteoblasts synthesize RANKL (receptor activator of NF-kappaB ligand), necessary for osteoclast formation and function, and osteoprotegerin (OPG), its decoy receptor. To investigate the effects of estrogen on the expression of OPG, RANKL, and ERs in human osteoblasts, cells were cultured with physiological (10(-10) M) and high-dose (10(-7) M) 17beta-estradiol for 24 and 48 h. Proteins and corresponding mRNA levels were quantitatively determined by immunocytochemistry and RT-PCR. OPG expression was significantly increased three- and sevenfold at 24 h with 10(-10) M (P < 0.05) and 10(-7) M (P < 0.01) estradiol, respectively, compared to untreated cells. Similar but smaller increases were seen at 48 h (P < 0.05). Osteoblasts treated with estradiol demonstrated increased RANKL protein expression at 24 h (P < 0.05), but this was not maintained at 48 h. ERalpha expression was significantly increased by high-dose estradiol (P < 0.01) at 24 h and dose-dependently increased at 48 h (P < 0.01), while ERbeta was only increased at 24 h (P < 0.01). The estrogen-induced protein expression of ER, OPG, and RANKL was abrogated when cells were cultured in the presence of the estrogen antagonist ICI 182780. mRNA levels at 24 h demonstrated a significant suppression of RANKL with the low-dose but not the high dose. ERalpha mRNA but not ERbeta expression was up-regulated by estrogen. Our results suggest that estrogen may exert its anti-resorptive effects on bone, at least in part, by stimulating ER and OPG expression in osteoblasts.

The expression of thyroid hormone receptors in human bone

The mechanism of action of thyroid hormones on bone is poorly understood. Thyroid hormones may act on bone cells either indirectly by increasing secretion of growth hormone (GH) and insulin-like growth factor-1 (IGF-1), or directly by influencing target genes via specific nuclear receptors. The presence of thyroid hormone receptors (TRs) has been demonstrated in human and rodent osteoblast-like cells and cell lines and recently in osteoclasts derived from an osteoclastoma in vitro. However, their presence in human bone in situ has not been reported. We have used specific polyclonal antibodies to TR-alpha 1, -alpha 2, and -beta 1 to investigate the expression of these receptors in sections of human osteophytes and heterotopic bone. Osteoblasts and osteoclasts were identified by alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), respectively, whereas chondrocytes were identified morphologically. At sites of endochondral and intramembranous bone formation, TR-beta 1 and the splice variant -alpha 2 were widely expressed by proliferating, mature, and hypertrophic chondrocytes and also in cells within the fibrous tissue and at the bone forming surfaces, respectively. They were also detected in osteoblasts, osteoclasts, and a few osteocytes at sites of bone remodeling. In contrast, TR-alpha 1 was the least expressed and was present mainly in osteoblasts at remodeling sites and in a few mature and undifferentiated chondrocytes. Our results show, for the first time, the presence and distribution of TRs in human bone in situ and suggest that the skeletal actions of thyroid hormones may be mediated via these receptors. Further studies are required to define the role of the individual receptor isoforms in bone metabolism.

Immunolocalisation of vascular endothelial growth factor (VEGF) in human neonatal growth plate cartilage.

Angiogenesis is essential for the replacement of cartilage by bone during growth and repair. In order to obtain a better understanding of the mechanisms regulating vascular invasion at sites of endochondral ossification we have investigated the expression of the endothelial cell‐specific mitogen, vascular endothelial growth factor (VEGF), by chondrocytes in human neonatal growth plates. VEGF was absent from chondrocytes in the resting zone and only weakly expressed by occasional chondrocytes in the proliferating region. In the hypertrophic zone the number of chondrocytes stained and the intensity of staining for VEGF increased with chondrocyte hypertrophy, maximum expression of VEGF being observed in chondrocytes in the lower hypertrophic and mineralised regions of the cartilage. These observations provide the first demonstration of the presence of VEGF in situ in developing human bone and are consistent with in vitro observations demonstrating the upregulation of proangiogenic growth factor production with increasing chondrocyte hypertrophy. The presence of numerous small blood vessels and vascular structures in the subchondral region where VEGF expression was maximal indicates that VEGF produced by hypertrophic chondrocytes may play a key role in the regulation of vascular invasion of the growth plate.

Expression and distribution of transforming growth factor-β isoforms and their signaling receptors in growing human bone

Transforming growth factors type beta (TGF-beta1, -beta2, and -beta3) are potent stimulators of bone formation and have been shown to regulate chondrocyte, osteoblast, and osteoclast formation and function. However, the distribution of the different isoforms and their signaling receptors in human bone in vivo has not previously been reported. Using samples of normal (neonatal rib) and pathological (osteophytic) developing human bone, we have investigated the expression of the different TGF-beta isoforms and their signaling receptors (TGF-betaRI and RII) at the messenger ribonucleic acid (mRNA) and protein levels by in situ hybridization and immunolocalization to establish the sites of TGF-beta production and their possible sites of action during human bone development in vivo. All three TGF-beta isoforms and the receptors were detected at sites of endochondral and intramembranous ossification. At sites of endochondral ossification, TGF-beta2 was detected in all zones of the cartilage, with the highest expression seen in the hypertrophic and mineralizing zones. TGF-beta3 was detected in proliferative and hypertrophic zone chondrocytes, while TGF-beta1 expression was restricted to the proliferative and upper hypertrophic zones. TGF-betaRI and RII exhibited similar distributions with maximum expression in the hypertrophic and mineralizing zones in the neonatal rib but in the resting/proliferative zone in the developing osteophyte. At sites of intramembranous ossification TGF-beta3 was the most widely distributed isoform and showed both matrix- and cell-associated staining. TGF-beta2 and -beta1 were expressed almost exclusively at sites of mineralization. These observations demonstrate that the different TGF-beta isoforms and their receptors exhibit distinct but overlapping patterns of expression, and support the hypothesis that they are involved in the regulation of endochondral and intramembranous ossification during human bone development in vivo.

Stromelysin-1 (MMP-3) and Stromelysin-2 (MMP-10) Expression in Developing Human Bone: Potential Roles in Skeletal Development

Stromelysin, a member of the matrix metalloproteinase family, demonstrates wide substrate specificity with the ability to degrade proteoglycan, fibronectin, laminin, casein, and the nonhelical region of collagen. The two forms of stromelysin (SL), types 1 (MMP-3) and 2 (MMP-10), share 82% sequence homology, but exhibit differences in cellular synthesis and inducibility by cytokines and growth factors in vitro. However, the distribution of the two isoforms in bone has not been reported. We investigated the presence of SL-1 and SL-2 in human osteophytic and neonatal rib bone using immunohistochemistry and, combined with a new method of in situ zymography, determined the activity of the immunolocalized stromelysins. Latent SL-1 was strongly expressed in the extracellular matrix in fibrous tissue surrounding areas of endochondral ossification in osteophytes, and adjacent to the periosteum of fetal rib bone. Active SL-1 expression was detected in osteocytes and the matrix surrounding osteocytic lacunae. SL-2 showed intense cell-associated staining at sites of resorption in areas of endochondral ossification and in resorptive cells at the chondro-osseous junction, which correlated with enzyme activity detected by zymography. Within the rib, active SL-2 expression was localized in chondrocytes of the growth plate, whereas only occasional SL-1 signal was evident. Vascular areas showed strong SL-2 staining with some proteolytic activity. SL-2, but not SL-1, was strongly expressed in osteoclasts and most mononuclear cells within the marrow. At sites of bone formation both isoforms were expressed by osteoblasts with SL-1 also present in osteoid. These results demonstrate, for the first time, the differential expression of SL-1 and SL-2 in developing human bone, indicating specific roles for the two isoforms. In situ zymography demonstrates that SL-2 is produced in an active form with associated degradation, whereas SL-1, in a matrix-bound proenzyme form, may act as a reservoir for later activation.

Tie2 ligands angiopoietin-1 and angiopoietin-2 are coexpressed with vascular endothelial cell growth factor in growing human bone.

Angiogenesis is essential for bone growth and repair. Recent studies have shown that the endothelial-specific mitogen vascular endothelial growth factor (VEGF) is a key regulator of vascular invasion into the growth plate in infant and adolescent animals. In order to identify mechanisms regulating VEGF-induced angiogenesis in growing bone, we have investigated the expression of the angiopoietins (Ang-1 and Ang-2) in human neonatal ribs. Ang-1 and Ang-2 exhibited similar patterns of staining in the growing rib. In the cartilage, expression of Ang-1 and Ang-2 increased with chondrocyte maturation. Ang-1, Ang-2, and VEGF were not detected in the resting zone except adjacent to vascular canals, and maximum expression was detected at the cartilage bone interface. In the cartilage, Ang-2 was more highly expressed than Ang-1 or VEGF, with staining observed in the proliferating, hypertrophic, and mineralized zones. In the bone, Ang-1, Ang-2, and VEGF were detected in modeling and remodeling sites. Ang-1 was detected in the majority of osteoblasts, osteoclasts, and in some marrow space cells. Ang-2 was expressed at variable levels by osteoblasts and osteoclasts in modeling and remodeling bone. VEGF was detected in cells at bone surfaces and in the marrow spaces. Strong staining for VEGF was observed in osteoblasts and osteoclasts in modeling and remodeling bone. In the perichondrium, Ang-1, Ang-2, and VEGF were most highly expressed adjacent to the hypertrophic zone and at sites of bone collar formation. In the periosteum, Ang-1, Ang-2, and VEGF expression colocalized with alkaline phosphatase expression. These observations provide the first evidence for the expression of the angiopoietins in growing human bone in vivo. The distribution of Ang-1, Ang-2, and VEGF indicate these factors may play key roles in the regulation of angiogenesis at sites of endochondral ossification, intramembranous ossification, and bone turnover in the growing human skeleton.

Colocalization of Glucocorticoid and Mineralocorticoid Receptors in Human Bone

Osteoporosis is a poorly understood but common complication of glucocorticoid therapy. The actions of glucocorticoids are mediated via glucocorticoid receptors (GRs), but in vitro, glucocorticoids also can bind to mineralocorticoid receptors (MRs). It is not known if MR protein is present in human bone and little is known of GR isoform expression (GRα and GRβ). GR and MR protein expression and possible sites of action were investigated in neonatal rib and adult iliac crest biopsy specimens using antibodies specific for MR, GRα, and GRαβ. Colocalization [MR GRα] [MR GRαβ] was performed using fluorescent‐conjugated secondary antibodies. GRα, GRβ, and MR show distinct but overlapping patterns of expression, suggesting important functions for each receptor type. Osteoclasts showed no staining for GRα but strong staining for GRαβ, indicating expression of GRβ and a specific role in addition to antagonizing the transcriptional activity of GRα. MR also was observed in osteoclasts and colocalized with GRαβ. Coexpression of MR, GRα, and GRαβ was seen in osteoblasts. Reverse‐transcription‐polymerase chain reaction (RT‐PCR) of cultured osteoblast RNA confirmed expression of both GRα and GRβ. Osteocytes stained with MR, GRα, and GRαβ antibodies but to a lesser degree than osteoblasts. In the neonatal rib cartilage, staining for GRα, GRαβ, and MR was present in approximately one‐half of the resting and hypertrophic chondrocytes and in most of proliferating chondrocytes and chondrocytes within the mineralizing matrix. Identification of MR raises the possibility that the physiological and pharmacologic effects of glucocorticoids on bone may be mediated via MR as well as GR and that GRα, GRβ, and MR synergize to influence corticosteroid metabolism in human bone.

Vacuolar H+-ATPase d2 subunit: molecular characterization, developmental regulation, and localization to specialized proton pumps in kidney and bone.

The ubiquitous multisubunit vacuolar-type proton pump (H+- or V-ATPase) is essential for acidification of diverse intracellular compartments. It is also present in specialized forms at the plasma membrane of intercalated cells in the distal nephron, where it is required for urine acidification, and in osteoclasts, playing an important role in bone resorption by acid secretion across the ruffled border membrane. It was reported previously that, in human, several of the renal pumps constituent subunits are encoded by genes that are different from those that are ubiquitously expressed. These paralogous proteins may be important in differential functions, targeting or regulation of H+-ATPases. They include the d subunit, where d1 is ubiquitous whereas d2 has a limited tissue expression. This article reports on an investigation of d2. It was first confirmed that in mouse, as in human, kidney and bone are two of the main sites of d2 mRNA expression. d2 mRNA and protein appear later during nephrogenesis than does the ubiquitously expressed E1 subunit. Mouse nephron-segment reverse transcription-PCR revealed detectable mRNA in all segments except thin limb of Henles loop and distal convoluted tubule. However, with the use of a novel d2-specific antibody, high-intensity d2 staining was observed only in intercalated cells of the collecting duct in fresh-frozen human kidney, where it co-localized with the a4 subunit in the characteristic plasma membrane-enhanced pattern. In human bone, d2 co-localized with the a3 subunit in osteoclasts. This different subunit association in different tissues emphasizes the possibility of the H+-ATPase as a future therapeutic target.

Distribution of platelet-derived growth factor (PDGF) a chain mRNA, protein, and PDGF-α receptor in rapidly forming human bone

Platelet-derived growth factors (PDGFs) are potent bone cell mitogens which stimulate the proliferation of osteoblastic cells, may also be involved in the regulation of osteoclastic bone resorption, and indirectly induce vascular endothelial cell proliferation and angiogenesis. In view of the established relationship between angiogenesis and osteogenesis, the production of PDGFs by both osteoblastic and vascular endothelial cells suggests that they may play a role in bone formation during skeletal development. We have used two human models of rapid bone formation, heterotopic bone and osteophytic bone, to investigate the expression of PDGF-A mRNA and protein and the PDGF-alpha receptor protein in vivo using in situ hybridization and immunohistochemistry. PDGF-A mRNA and protein were widely distributed throughout heterotopic and osteophytic bone. Within the cartilaginous tissue PDGF-A mRNA and protein were most strongly expressed by mature chondrocytes with decreased expression in the hypertrophic zone and almost no staining in the mineralizing and mineralized zones. PDGF mRNA and protein were also expressed in cells of small blood vessels within fibrous and cartilaginous tissue. In contrast, PDGF-alpha receptor expression was restricted to a minority of hypertrophic chondrocytes and sites of vascular invasion. Within the bone and fibrous tissue the growth factor and the receptor were widely distributed, being detected on most cells at sites of bone formation or in remodeling sites; no receptor was detected on osteoclasts. These data demonstrate the widespread expression of PDGF-A and its receptor in forming human bone and indicate that this growth factor may exert autocrine and paracrine effects to regulate osteogenesis during skeletal development.

Distribution of matrix metalloproteinases and their inhibitor, TIMP-1, in developing human osteophytic bone

Connective tissues synthesise and secrete a family of matrix metalloproteinases (MMPs) which are capable of degrading most components of the extracellular matrix. Animal studies suggest that the MMPs play a role in bone turnover. Using specific polyclonal antisera, immunohistochemistry was used to determine the patterns of synthesis and distribution of collagenase (MMP‐1), stromelysin (MMP‐3), gelatinase A (MMP‐2) and gelatinase B (MMP‐9) and of the tissue inhibitor of metalloproteinases‐1 (TIMP‐1) within developing human osteophytic bone. The different MMPs and TIMP showed distinct patterns of localisation. Collagenase expression was seen at sites of vascular invasion, in osteoblasts synthesising new matrix and in some osteoclasts at sites of resorption. Chondrocytes demonstrated variable levels of collagenase and stromelysin expression throughout the proliferative and hypertrophic regions, stromelysin showing both cell‐associated and strong matrix staining. Intense gelatinase B expression was observed at sites of bone resorption in osteoclasts and mononuclear cells. Gelatinase A was only weakly expressed in the fibrocartilage adjacent to areas of endochondral ossification. There was widespread but variable expression of TIMP‐1 throughout the fibrous tissue, cartilage and bone. These results indicate that MMPs play a role in the development of human bone from cartilage and fibrous tissue and are likely to have multiple functions.