Anne M. Delany

Mechanisms of Glucocorticoid Action in Bone

Abstract: Glucocorticoids cause profound effects on bone cell replication, differentiation, and function. Glucocorticoids increase bone resorption by stimulating osteoclastogenesis by increasing the expression of RANK ligand and decreasing the expression of its decoy receptor, osteoprotegerin. In accordance with the increase in bone resorption, glucocorticoids stimulate the expression of collagenase 3 by posttranscriptional mechanisms. The most significant effect of glucocorticoids in bone is an inhibition of bone formation. This is because of a decrease in the number of osteoblasts and their function. The decrease in cell number is secondary to a decrease in osteoblastic cell replication and differentiation, and an increase in the apoptosis of mature osteoblasts. Glucocorticoids decrease osteoblastic function directly and indirectly through the modulation of growth factor expression, receptor binding, or binding protein levels. Clinically, patients with glucocorticoid‐induced osteoporosis (GIOP) develop bone loss in the first few months of glucocorticoid exposure, and modest doses of glucocorticoids increase the risk of fractures of the spine and hip. Bisphosphonates inhibit bone resorption and prevent and revert the bone loss that follows glucocorticoid exposure. Anabolic agents, such as parathyroid hormone, stimulate bone formation and can increase bone mass in GIOP.

miR-29 modulates WNT signaling in human osteoblasts through a positive feedback loop

Differentiation of human mesenchymal stem cells into osteoblasts is controlled by extracellular cues. Canonical Wnt signaling is particularly important for maintenance of bone mass in humans. Post-transcriptional regulation of gene expression, mediated by microRNAs, plays an essential role in the control of osteoblast differentiation. Here, we find that miR-29a is necessary for human osteoblast differentiation, and miR-29a is increased during differentiation in the mesenchymal precursor cell line hFOB1.19 and in primary cultures of human osteoblasts. Furthermore, the promoter of the expressed sequence tag containing the human miR-29a gene is induced by canonical Wnt signaling. This effect is mediated, at least in part, by two T-cell factor/LEF-binding sites within the proximal promoter. Furthermore, we show that the negative regulators of Wnt signaling, Dikkopf-1 (Dkk1), Kremen2, and secreted frizzled related protein 2 (sFRP2), are direct targets of miR-29a. Endogenous protein levels for these Wnt antagonists are increased in cells transfected with synthetic miR-29a inhibitor. In contrast, transfection with miR-29a mimic decreases expression of these antagonists and potentiates Wnt signaling. Overall, we demonstrate that miR-29 and Wnt signaling are involved in a regulatory circuit that can modulate osteoblast differentiation. Specifically, canonical Wnt signaling induces miR-29a transcription. The subsequent down-regulation of key Wnt signaling antagonists, Dkk1, Kremen2, and sFRP2, by miR-29a potentiates Wnt signaling, contributing to a gene expression program important for osteoblast differentiation. This novel regulatory circuit provides additional insight into how microRNAs interact with signaling molecules during osteoblast differentiation, allowing for fine-tuning of intricate cellular processes.

Osteopenia and decreased bone formation in osteonectin-deficient mice

Bone continuously remodels in response to mechanical and physiological stresses, allowing vertebrates to renew bone as adults. Bone remodeling consists of the cycled synthesis and resorption of collagenous and noncollagenous extracellular matrix proteins, and an imbalance in this process can lead to disease states such as osteoporosis, or more rarely, osteopetrosis. There is evidence that the extracellular matrix glycoprotein osteonectin or secreted protein acidic and rich in cysteine (BM-40) may be important in bone remodeling. Osteonectin is abundant in bone and is expressed in areas of active remodeling outside the skeleton. In vitro studies indicate that osteonectin can bind collagen and regulate angiogenesis, metalloproteinase expression, cell proliferation, and cell-matrix interactions. In some osteopenic states, such as osteogenesis imperfecta and selected animal models for bone fragility, osteonectin expression is decreased. To determine the function of osteonectin in bone, we used contact x-ray, histomorphometry, and Northern blot analysis to characterize the skeletal phenotype of osteonectin-null mice. We found that osteonectin-null mice have decreased bone formation and decreased osteoblast and osteoclast surface and number, leading to decreased bone remodeling with a negative bone balance and causing profound osteopenia. These data indicate that osteonectin supports bone remodeling and the maintenance of bone mass in vertebrates.

miR‐29 suppression of osteonectin in osteoblasts: Regulation during differentiation and by canonical Wnt signaling

The matricellular protein osteonectin, secreted protein acidic and rich in cysteine (SPARC, BM‐40), is the most abundant non‐collagenous matrix protein in bone. Matricellular proteins play a fundamental role in the skeleton as regulators of bone remodeling. In the skeleton, osteonectin is essential for the maintenance of bone mass and for balancing bone formation and resorption in response to parathyroid hormone (PTH). It promotes osteoblast differentiation and cell survival. Mechanisms regulating the expression of osteonectin in the skeleton and in other tissues remain poorly understood. We found that the proximal region of the mouse osteonectin 3′ untranslated region (UTR) contains a well‐conserved, dominant regulatory motif that interacts with microRNAs (miRs)‐29a and ‐29c. Transfection of osteoblastic cells with miR‐29a inhibitors increased osteonectin protein levels, whereas transfection of miR‐29a precursor RNA decreased osteonectin. miR‐29a and ‐29c were increased during osteoblastic differentiation in vitro. The up‐regulation of these miRNAs correlated with decreased osteonectin protein during the matrix maturation and mineralization phases of late differentiation. In contrast, osteonectin transcript levels remained relatively constant during this process, implying repression of translation. Treatment of osteoblasts with LiCl induced miR‐29a and ‐29c expression and decreased osteonectin synthesis. When cells were treated with Dickkopf‐1 (Dkk‐1), miR‐29a and ‐29c expression was repressed. These data suggest that canonical Wnt signaling, which is increased during osteoblastic differentiation, induces expression of miR‐29. Osteonectin and miR‐29 are co‐expressed in extra‐skeletal tissues, and the post‐transcriptional mechanisms regulating osteonectin in osteoblasts are likely to be active in other cell systems. J. Cell. Biochem. 108: 216–224, 2009.

Cortisol inhibits the differentiation and apoptosis of osteoblasts in culture

Glucocorticoids decrease the replication of cells of the osteoblastic lineage and the function of the osteoblast. However, under certain conditions, they enhance the differentiation of osteoblastic cells, an effect that appears contradictory to their inhibitory actions on cell function. In this study we examine the effects of cortisol on the proliferation, differentiation, and fate of osteoblastic enriched cells from 22-day-old fetal rat calvariae (osteoblastic cells) in the absence and presence of beta-glycerophosphate. In the absence of beta-glycerophosphate, there was a progressive accumulation of DNA and cells, which was impaired by cortisol. In the presence of beta-glycerophosphate, there was an initial accumulation of DNA and cells followed by a marked decline that was prevented by cortisol. Despite the sustained number of cells, cortisol did not affect their mineralization, and inhibited Core binding factor a1 (Cbfa1), but not alkaline phosphatase, osteocalcin, or type I collagen transcripts. The decrease in cell number by cortisol observed in the absence of beta-glycerophosphate was due to a decrease in DNA synthesis, whereas the increase in cell number observed in the presence of beta-glycerophosphate was due to a relative increase in DNA synthesis and a decrease in apoptosis as determined by DNA fragmentation and acridine orange staining of the cells. This was correlated by a decrease in transcripts of proapoptotic genes and caspase 3 activity, and an increase of antiapoptotic genes. In conclusion, cortisol decreases the replication of cells of the osteoblastic lineage, but under conditions of differentiation/mineralization, cortisol prevents terminal differentiation of the cells and maintains an immature cell population.

Effects of cortisol and bone morphogenetic protein-2 on stromal cell differentiation: correlation with CCAAT-enhancer binding protein expression.

Bone marrow stroma contain pluripotential cells with the potential to differentiate into various mesenchymal cell lineages. We compared the effect of cortisol and bone morphogenetic protein-2 (BMP-2) on the differentiation of murine ST-2 stromal cells into mature osteoblasts or adipocytes. ST-2 cells were cultured for 3-27 days in the presence of 10% fetal bovine serum, 100 microg/mL ascorbic acid, and 5 mmol/L beta-glycerolphosphate in the presence or absence of cortisol at 1 micromol/L or BMP-2 at 1 nmol/L. Untreated ST-2 cells expressed high levels of alkaline phosphatase activity (APA) 15 days after confluence, and this was followed by the appearance of mineralized nodules after 24 days. BMP-2 accelerated and intensified the appearance of cells expressing APA and the presence of mineralized nodules. In contrast, cortisol decreased APA, prevented the formation of mineralized nodules, and induced a cellular phenotype characteristic of adipocytes. Untreated stromal cells expressed osteocalcin, Cbfa1, type I collagen, and alkaline phosphatase mRNA. BMP-2 increased osteocalcin and alkaline phosphatase mRNA, whereas cortisol suppressed their expression, as well as Cbfa1 and type I collagen transcripts. Cortisol enhanced, and BMP-2 downregulated, peroxisome proliferator-activated receptor gamma 2 and adipsin transcripts. The C/EBP transcription factors regulate genes critical for adipocytic and osteoblastic differentiation. Cortisol increased the expression of C/EBP alpha, beta, delta, and gamma mRNA levels, whereas BMP-2 had minor effects on C/EBP expression. In conclusion, BMP-2 accelerates the differentiation of stromal cells toward an osteoblastic phenotype, whereas glucocorticoids induce their differentiation toward an adipocytic phenotype.

MicroRNA biogenesis and regulation of bone remodeling

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression. This review will highlight our current understanding of miRNA biogenesis and mechanisms of action, and will summarize recent work on the role of miRNAs, including the miR-29 family, in bone remodeling. These studies represent the first steps in demonstrating the importance of miRNAs in the control of osteoblast and osteoclast differentiation and function. An in-depth understanding of the roles of these regulatory RNAs in the skeleton will be critical for the development of new therapeutics aimed at treating bone loss and perhaps facilitating fracture repair.

Infrared Analysis of the Mineral and Matrix in Bones of Osteonectin‐Null Mice and Their Wildtype Controls

Osteonectin function in bone was investigated by infrared analysis of bones from osteonectin‐null (KO) and wildtype mice (four each at 11, 17, and 36 weeks). An increase in mineral content and crystallinity in newly formed KO bone and collagen maturity at all sites was found using FTIR microspectroscopy and imaging; consistent with osteonectins postulated role in regulating bone formation and remodeling.

CORTISOL INCREASES INTERSTITIAL COLLAGENASE EXPRESSION IN OSTEOBLASTS BY POST-TRANSCRIPTIONAL MECHANISMS

Glucocorticoids regulate both bone formation and bone resorption. In osteoblasts, they inhibit type I collagen synthesis; however, there is limited information about their effects on interstitial collagenase, the enzyme that degrades type I collagen. We used primary cultures of osteoblast-enriched cells from fetal rat calvariae (Ob cells) to study the effects of cortisol on collagenase expression. Northern blot analysis showed that cortisol increased collagenase transcript levels in a dose- and time-dependent manner, which was paralleled by an increase in immunoreactive metalloproteinase in the culture medium. Cortisol increased the half-life of collagenase mRNA from 6 to 12 h in transcription-arrested Ob cells. In contrast, cortisol modestly decreased collagenase gene transcription after 24 h of treatment. The up-regulation of collagenase by cortisol is osteoblast-specific, since the glucocorticoid decreased phorbol 12-myristate 13-acetate-induced collagenase mRNA expression in rat fibroblasts, a result that agrees with other studies of collagenase gene regulation in fibroblastic cells. In conclusion, cortisol increases interstitial collagenase transcript levels by post-transcriptional mechanisms in osteoblastic cells. Our data demonstrate that glucocorticoids regulate collagenase gene expression in a novel tissue-specific manner, further highlighting the differences in gene regulation between osteoblastic and fibroblastic cells.

Cortisol Inhibits the Synthesis of Insulin-like Growth Factor-binding Protein-5 in Bone Cell Cultures by Transcriptional Mechanisms

Glucocorticoids inhibit the synthesis of insulin-like growth factor-binding protein-5 (IGFBP-5) in osteoblasts, but the mechanisms involved are unknown. IGFBP-5 stimulates bone cell growth, and its inhibition by glucocorticoids may be relevant to the action of this binding protein on bone formation. We tested the effects of cortisol on IGFBP-5 expression in cultures of osteoblast-enriched cells from fetal rat calvariae (Ob cells). Cortisol decreased IGFBP-5 polypeptide levels in the extracellular matrix and caused a time- and dose-dependent decrease in IGFBP-5 mRNA. IGFBP-5 transcripts were markedly decreased by cycloheximide, and further suppressive effects of cortisol could not be determined. Cortisol did not modify the decay of IGFBP-5 mRNA in transcriptionally arrested Ob cells. Cortisol decreased IGFBP-5 hnRNA, the rate of IGFBP-5 transcription, and the activity of the murine IGFBP-5 promoter by 35% in transient transfection experiments. Deletion analysis showed that the region responsive to cortisol is from base pairs −70 to +22, and E-box-binding proteins or c-Myb-related nuclear factors may be involved in its regulation. In conclusion, cortisol inhibits IGFBP-5 transcription in Ob cells through the Myb-binding domain. This effect may be partly responsible for the effect of glucocorticoids on bone formation.