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Biomedical subjects

Anne M Delany

Publications and source records attributed to Anne M Delany.

10 recordsLinked to original sources
PubMed2007

Increased Notch 1 expression and attenuated stimulatory G protein coupling to adenylyl cyclase in osteonectin-null osteoblasts.

Osteonectin, or secreted protein acidic and rich in cysteine, is one of the most abundant noncollagen matrix components in bone. This matricellular protein regulates extracellular matrix assembly and maturation in addition to modulating cell behavior. Mice lacking osteonectin develop severe low-turnover osteopenia, and in vitro studies of osteonectin-null osteoblastic cells showed that osteonectin supports osteoblast formation, maturation, and survival. The present studies demonstrate that osteonectin-null osteoblastic cells have increased expression of Notch 1, a well-documented regulator of cell fate in multiple systems. Furthermore, osteonectin-null cells are more plastic and less committed to osteoblastic differentiation, able to pursue adipogenic differentiation given the appropriate signals. Notch 1 transcripts are down-regulated by inducers of cAMP in both wild-type and osteonectin-null osteoblasts, suggesting that the mutant osteoblasts may have a defect in generation of cAMP in response to stimuli. Indeed, many bone anabolic agents signal through increased cAMP. Wild-type and osteonectin-null osteoblasts generated comparable amounts of cAMP in response to forskolin, a direct stimulator of adenylyl cyclase. However, the ability of osteonectin-null osteoblasts to generate cAMP in response to cholera toxin, a direct stimulator of Gs, was attenuated. These data imply that osteonectin-null osteoblasts have decreased coupling of Gs to adenylyl cyclase. Because osteonectin promotes G protein coupling to an effector, our studies support the concept that low-turnover osteopenia can result from reducing G protein coupled receptor activity.

Adenylyl Cyclases↗
PubMed2003

CCAAT/enhancer binding protein homologous protein (DDIT3) induces osteoblastic cell differentiation.

CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP/DDIT3), a member of the C/EBP family of transcription factors, plays a role in cell survival and differentiation. CHOP/DDIT3 binds to C/EBPs to form heterodimers that do not bind to consensus Cebp sequences, acting as a dominant-negative inhibitor. CHOP/DDIT3 blocks adipogenesis, and we postulated it could induce osteoblastogenesis. We investigated the effects of constitutive CHOP/DDIT3 overexpression in murine ST-2 stromal cells transduced with retroviral vectors. ST-2 cells differentiated toward osteoblasts, and CHOP/DDIT3 accelerated and enhanced the appearance of mineralized nodules, and the expression of osteocalcin and alkaline phosphatase mRNAs, particularly in the presence of bone morphogenetic protein-2. CHOP/DDIT3 overexpression opposed adipogenesis, and did not cause substantial changes in cell number. CHOP/DDIT3 overexpression did not modify C/EBPalpha or -beta mRNA levels but decreased C/EBPdelta after 24 d of culture. Electrophoretic mobility shift and supershift assays demonstrated that overexpression of CHOP/DDIT3 decreased the binding of C/EBPs to their consensus sequence by interacting with C/EBPalpha and -beta, confirming its dominant-negative role. In addition, CHOP/DDIT3 enhanced bone morphogenetic protein-2/Smad signaling. In conclusion, CHOP/DDIT3 enhances osteoblastic differentiation of stromal cells, in part by interacting with C/EBPalpha and -beta and also by enhancing Smad signaling.

Alkaline Phosphatase↗
PubMed2003

AU-rich elements in the collagenase 3 mRNA mediate stabilization of the transcript by cortisol in osteoblasts.

Collagenase 3 degrades collagen fibrils and is necessary for bone resorption. Cortisol increases collagenase 3 mRNA in osteoblasts by stabilizing collagenase 3 transcripts. To understand mechanisms involved, we used RNA electrophoretic mobility shift assay and RNA turnover studies. Cortisol increased the binding of Ob cell cytosolic extracts to AU-rich sequences in the collagenase 3 3'-untranslated region (UTR). No cortisol-dependent protein complexes were formed with the coding region or the 5'-UTR. Functional assays, using transient transfections of CMV-driven c-fos collagenase 3'-UTR chimeric constructs, demonstrated that the 3'-UTR of collagenase 3 stabilizes c-fos mRNA in transcriptionally arrested Ob cells, cortisol prolongs the transcript half-life, and mutations of AU-rich sequences destabilize c-fos transcripts precluding the cortisol effect. Purification of osteoblast cytosolic extracts by ultracentrifugation, ion exchange, and RNA affinity chromatography, and polyacrylamide gel electrophoresis followed by mass spectroscopy identified specific proteins. RNA gel mobility supershift assays demonstrated that vinculin and far upstream element (FUSE)-binding protein 2 interacted with collagenase 3 3'-UTR sequences, and RNA interference demonstrated these proteins altered collagenase mRNA stability. In conclusion, AU-rich sequences of the 3'-UTR of collagenase 3 and vinculin and FUSE-binding protein 2 regulate collagenase mRNA stability in osteoblasts.

3' Untranslated Regions↗
PubMed2003

Notch 1 impairs osteoblastic cell differentiation.

Notch receptors are single pass transmembrane receptors activated by membrane-bound ligands with a role in cell proliferation and differentiation. As Notch 1 and 2 mRNAs are expressed by osteoblasts and induced by cortisol, we postulated that Notch could regulate osteoblastogenesis. We investigated the effects of retroviral vectors directing the constitutive expression of the Notch 1 intracellular domain (NotchIC) in murine ST-2 stromal and in MC3T3 cells. NotchIC overexpression was documented by increased Notch 1 transcripts and activity of the Notch-dependent Hairy Enhancer of Split promoter. In the presence of bone morphogenetic protein-2 (BMP-2), ST-2 cells differentiated toward osteoblasts forming mineralized nodules, and Notch 1 opposed this effect and decreased the expression of osteocalcin, type I collagen, and alkaline phosphatase transcripts and Delta2Delta FosB protein. Further, NotchIC decreased Wnt/beta-catenin signaling. As cells differentiated in the presence of BMP-2, they underwent apoptosis, and Notch opposed this event. In the presence of cortisol, NotchIC induced the formation of mature adipocytes and enhanced the effect of cortisol on adipsin, peroxisome proliferator-activated receptor-gamma2 and CCAAT enhancer binding protein alpha and delta mRNA levels. NotchIC also opposed MC3T3 cell differentiation and the expression of a mature osteoblastic phenotype. In conclusion, NotchIC impairs osteoblast differentiation and enhances adipogenesis in stromal cell cultures.

3T3 Cells↗
PubMed2003

Osteonectin-null mutation compromises osteoblast formation, maturation, and survival.

Osteonectin, also known as SPARC (secreted protein acidic and rich in cysteine) or BM-40, is one of the most abundant noncollagenous proteins in bone. Analysis of osteonectin-null mice revealed that osteonectin is necessary for the maintenance of bone mass and normal remodeling, as osteonectin-null mice have decreased osteoblast number and bone formation rate. Cultures of bone marrow stromal cells and osteoblasts from control and osteonectin-null mice were used to determine the cellular basis for the mutant phenotype. We found that marrow stroma from osteonectin-null mice contains fewer osteoblastic precursors than that of control mice, and the osteonectin-null mutation did not affect the proliferation rate of stromal cells or osteoblasts. Whereas osteonectin-null cells could adopt an osteoblastic phenotype, a smaller proportion of these cells expressed markers of a fully differentiated osteoblast. Mutant cells exhibited decreased formation of mineralized nodules, as well as diminished expression of osteocalcin mRNA and response to PTH. Furthermore, osteonectin-null cells showed an increased tendency to form adipocytes, with enhanced expression of the adipocytic markers adipsin and CCAAT/enhancer binding protein delta. Osteonectin-null cells were also more susceptible to environmental stresses. These data indicate that osteonectin is important for osteoblast formation, maturation, and survival.

Animals↗
PubMed2003

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 osteonectin's postulated role in regulating bone formation and remodeling. Mineral and matrix properties of tibias of osteonectin-null mice and their age- and background-matched wildtype controls were compared using Fourier-transform infrared microspectroscopy (FTIRM) and infrared imaging (FTIRI) at 10- and 7-mm spatial resolution, respectively. The bones came from animals that were 11, 17, and 36 weeks of age. Individual FTIRM spectra were acquired from 20 x 20 microm areas, whereas 4096 simultaneous FTIRI spectra were acquired from 400 x 400 microm areas. The FTIRM data for mineral-to-matrix, mineral crystallinity, and collagen maturity were highly correlated with the FTIRI data in similar regions. In general, the osteonectin-null mice bones had higher mineral contents and greater crystallinity (crystal size and perfection) than the age-matched wildtype controls. Specifically, the mineral content of the newly forming periosteal bone was increased in the osteonectin-null mice; the crystallinity of the cortical bone was decreased in all but the oldest animals, relative to the wildtype. The most significant finding, however, was increased collagen maturity in both the cortical and trabecular bone of the osteonectin-null mice. These spectroscopic data are consistent with a mechanism of decreased bone formation and remodeling.

Animals↗
PubMed2002

Cortisol regulates the expression of Notch in osteoblasts.

Glucocorticoids have important effects on osteoblastic replication, differentiation, and function, and the Notch family of receptors is considered to play a role in osteoblastic cell differentiation. We postulated that cortisol could regulate Notch and Notch ligand expression in osteoblastic cells, providing an additional mechanism by which glucocorticoids could regulate osteoblastic differentiation. We examined the expression and regulation of Notch1, 2, 3, and 4 and their ligands Jagged 1 and 2 and Delta 1 and 3 by cortisol in cultures of osteoblastic MC3T3-E1 cells. Cortisol caused a time-dependent increase in Notch1 and 2 mRNA levels in MC3T3 cells. Notch3 and 4 were not detected in the presence or absence of cortisol. MC3T3 cells expressed Delta 1 and Jagged 1 but not Jagged 2 or Delta 3 mRNAs, and cortisol did not have a substantial effect on the expression of any of these ligands. Cortisol increased the rate of Notch1 and 2 transcription and, in transcriptionally arrested cells, did not modify the decay of the transcripts, indicating a transcriptional level of control. In conclusion, cortisol stimulates Notch1 and 2 transcription in osteoblasts. Since Notch signaling appears to play a negative role in osteoblastic differentiation, its increased expression could be relevant to the actions of cortisol in bone.

Animals↗
PubMed2002

Mechanisms of glucocorticoid action in bone.

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.

Animals↗
PubMed2002

Effects of glucocorticoids on the skeleton.

Glucocorticoids have profound effects on mineral metabolism and skeletal cell function, and an excess can lead to the development of short stature and osteoporosis. The most significant actions of glucocorticoids are on chondrocytes and bone-forming cells, where they act, directly and indirectly, and regulate the local synthesis of insulin-like growth factor-I.

Animals↗