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E Canalis

Publications and source records attributed to E Canalis.

At least 19 recordsLinked to original sources
PubMed2005

Signals that determine the fate of osteoblastic cells.

Signals that determine skeletal cell fate, function and apoptosis are critical to normal bone remodeling. Skeletal cells synthesize growth factors; some regulate cell replication, others regulate osteoblastic differentiation. Bone morphogenetic proteins (BMP) and Wnts induce the differentiation of mesenchymal cells toward mature osteoblasts. The action of BMPs is regulated by extracellular antagonists. These often act by binding to BMPs and preventing their binding to cell surface receptors. Wnt is essential for osteoblastogenesis, and mutations of Wnt co-receptors are associated with changes in bone mass. Wnt activity, like BMPs, is regulated by extracellular and intracellular antagonists. Notch, a family of transmembrane receptors, opposes Wnt signaling and inhibits osteoblastic differentiation. BMP, Wnt and Notch play a central role in osteoblastic cell fate.

Animals↗
PubMed2004

Overexpression of IGF-binding protein 5 alters mineral and matrix properties in mouse femora: an infrared imaging study.

The anabolic effects of insulin-like growth factors (IGFs) are modulated by a family of IGF-binding proteins (IGFBPs). Among the six known IGFBPs, IGFBP-5 is considered to play a role in bone formation. To investigate the effects of IGFBP-5 on bone mineral and matrix properties, femurs from transgenic mice overexpressing IGFBP-5 under the control of the osteocalcin promoter were evaluated by Fourier Transform Infrared Imaging (FTIRI). Analyses were done at the time of maximal osteocalcin expression (5 weeks). The spectroscopic parameters monitored were mineral-to-matrix ratio (indicative of the relative amount of mineral present), mineral crystallinity (index of the mineral crystal size and perfection) and collagen maturity (reflecting the ratio of non-reducible and reducible collagen cross-links). Multiple fields were selected for each femur, ranging from epiphysis to diaphysis. Previously, we showed that these transgenic mice display decreased osteoblastic function and osteopenia. In the present work, FTIRI showed that transgenic mice as compared to wild types have a different pattern of bone mineralization and matrix maturation. Specifically, cortical bone, primary spongiosa, and secondary ossification centers had lower values for mineral-to-matrix ratio and collagen maturity. Differences were not statistically significant in all cases although the trends were consistent. The mineral crystallinity did not vary significantly between the two groups, implying that the crystal maturation of mineral was not affected by IGFBP-5 overexpression. This study demonstrates that femurs from transgenic mice over expressing IGFBP-5 under the control of the osteocalcin promoter have modest alterations in mineral and matrix distribution, consistent with a role of IGF in osteoblast maturation.

Animals↗
PubMed2004

Overexpression of insulin-like growth factor binding protein-5 decreases osteoblastic function in vitro.

Skeletal cells synthesize insulin-like growth factors (IGF) and their binding proteins (IGFBP). The effects of IGFBP-5 on bone cell growth and osteoblastic function are controversial, and transgenic mice overexpressing IGFBP-5 exhibit osteopenia. The mechanisms were not explored, and in this study, we investigated the effects of IGFBP-5 overexpression in MC3T3 cells in vitro. MC3T3 cells were transduced with a retroviral vector (pLPCX) or a vector directing IGFBP-5 transcription under the control of a constitutive promoter. Untreated MC3T3 cells expressed alkaline phosphatase, and osteocalcin mRNA 1 week after confluence and at 4 weeks of culture they formed mineralized nodules. IGFBP-5 overexpression delayed the appearance of alkaline phosphatase and osteocalcin mRNA, decreased type I collagen and osteopontin mRNA and alkaline phosphatase activity (APA), and inhibited the formation of mineralized nodules. IGFBP-5 caused a modest stimulation of DNA synthesis. In conclusion, overexpression of IGFBP-5 decreases osteoblastic function possibly by binding IGFs in the bone microenvironment.

3T3 Cells↗
PubMed2004

Transgenic mice expressing selected insulin-like growth factor-binding protein-5 fragments do not exhibit enhanced bone formation.

Skeletal cells synthesize insulin like growth factors (IGF) and six IGF binding proteins (IGFBP). IGFBP-5 and fragments were reported to stimulate bone cell growth and parameters of osteoblastic function. We investigated the effects of IGFBP-5 1-162 and 1-193 on bone remodeling in transgenic mice overexpressing these fragments under the control of the osteocalcin promoter. Transgenic mice had normal appearance, weight, and bone mineral density. Static and dynamic histomorphometry revealed that transgenic mice overexpressing IGFBP-5 1-162 or 1-193 had normal trabecular bone volume, osteoblast and osteoclast number, and normal bone formation rate. MC3T3 cells transduced with retroviral vectors overexpressing IGFBP-5 1-235, 1-193, and 1-162 fragments displayed normal cell growth and maturation, and failed to enhance the expression of alkaline phosphatase, osteocalcin, and type I collagen mRNA when compared to cells transduced with vector alone. In conclusion, transgenic mice expressing IGFBP-5 1-162 and 1-193 in the bone microenvironment do not exhibit an obvious skeletal phenotype.

3T3 Cells↗
PubMed2003

Noggin arrests stromal cell differentiation in vitro.

Noggin is a glycoprotein that binds bone morphogenetic proteins (BMPs) selectively and, when added to osteoblasts, it opposes the effects of BMPs. However, the consequences of its continued expression in stromal cells are not known. We investigated the effects of noggin overexpression under the control of a constitutive promoter, on murine ST-2 stromal cells, and its impact on stromal cells from transgenic mice overexpressing noggin under the control of the osteocalcin promoter. ST-2 cells were transduced with a retroviral vector (pLPCX) or a vector driving noggin (pLPCX noggin). Untreated (pLPCX) ST-2 cells developed the appearance of mineralized nodules and expressed osteocalcin. pLPCX noggin delayed the appearance of mineralized nodules and prevented the expression of osteocalcin. Noggin also prevented the cortisol-dependent induction of peroxisome proliferator-activated receptor gamma2 and adipsin transcripts, indicating a generalized inhibition of cell differentiation. Primary stromal cells from noggin transgenic mice displayed impaired differentiation when compared to cells from wild-type animals and did not express osteocalcin mRNA. In conclusion, noggin arrests the differentiation of stromal cells, preventing cellular maturation.

Animals↗
PubMed2003

Skeletal overexpression of noggin results in osteopenia and reduced bone formation.

Skeletal cells synthesize bone morphogenetic proteins (BMPs) and BMP antagonists. Noggin is a glycoprotein that binds BMPs selectively and antagonizes BMP actions. Noggin expression in osteoblasts is induced by BMPs and noggin opposes the effects of BMPs on osteoblastic differentiation and function in vitro. However, its effects in vivo are not known. We investigated the direct in vivo effects of noggin on bone remodeling in transgenic mice overexpressing noggin under the control of the osteocalcin promoter. Noggin transgenics suffered long bone fractures in the first month of life. Total, vertebral, and femoral bone mineral densities were reduced by 23-29%. Static and dynamic histomorphometry of the femur revealed that noggin transgenic mice had decreased trabecular bone volume, number of trabeculae, and bone formation rate. Osteoblast surface and number of osteoblasts/trabecular area were not significantly decreased, indicating impaired osteoblastic function. Osteoclast surface and number were normal/decreased, there was no increase in bone resorption, and the tissue had the appearance of woven bone. Vertebral microcomputed tomography scanning confirmed decreased trabecular bone volume and trabecular number. In conclusion, transgenic mice overexpressing noggin in the bone microenvironment have decreased trabecular bone volume and impaired osteoblastic function, leading to osteopenia and fractures.

Animals↗
PubMed2002

Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts: comparison between rat type I collagen and rat osteocalcin promoters.

The osteocalcin (OC) and a 2.3 kb fragment of the collagen promoter (Col2.3) have been used to restrict transgenic expression of a variety of proteins to bone. Transgenic mice carrying a green fluorescent protein (GFP) gene driven by each promoter were generated. Strong GFP expression was detected in OC-GFP mice in a few osteoblastic cells lining the endosteal bone surface and in scattered osteocytes within the bone matrix in long bones from 1-day-old to 6-month-old transgenic animals. Similar findings were noted in the forming tooth in which only individual odontoblasts expressed GFP without detectable expression from the dental pulp. This limited pattern of OC-GFP-positive cells contrasts with the uniform expression in the Col2.3GFP mice in which large proportion of osteoblasts, odontoblasts, and osteocytes strongly expressed the transgene. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed. The activity of both transgenes was restricted to mineralized nodules but the number of positive cells was lower in the OC-GFP-derived cultures. The different temporal and spatial pattern of each transgene in vivo and in vitro reveals potential advantages and disadvantages of these two transgene models.

Animals↗
PubMed2002

Bronchial bacterial colonization in patients with resectable lung carcinoma.

The pattern and clinical implications of bronchial bacterial colonization have been widely investigated in patients with chronic lung disease, particularly chronic obstructive pulmonary disease. The main aim of this study was to determine the frequency and risk factors for bronchial colonization in lung cancer patients who have undergone surgical resection. Forty-one patients with resectable lung cancer (22 (54%) active smokers, 52+/-23 pack-yrs) with a mean forced expiratory volume in one second of 80+/-16% predicted, were studied with bilateral protected specimen brush and lung tissue biopsy during the surgical procedure. Quantitative bacterial culture, susceptibility tests and histological examination of samples were performed. Bronchial colonization with > or = 1 potential pathogenic micro-organism was found in 17 of 41 (41%) patients. The most frequent strains isolated were: Haemophilus influenzae (35%), Streptococcus pneumoniae (13%) and Pseudomonas spp. (9%). The risk factors for bronchial colonization were central location of the tumour (odds ratio (OR)=9.2, confidence interval (CI) 95%=2.1-39.6, p=1.003) and increased body mass index (OR=1.6, CI 95%=1.2-2.2, p=0.005). The frequency of postoperative infectious pulmonary complications was low (five cases (12%)) and no relationship was observed with bronchial colonization. Patients with resectable lung carcinoma had a high rate of bronchial colonization (41%), mainly with potential pathogenic microorganisms. The independent risk factors for colonization in these patients were central location of the tumour and a high body mass index.

Aged↗
PubMed2002

Transgenic mice overexpressing insulin-like growth factor binding protein-5 display transiently decreased osteoblastic function and osteopenia.

Skeletal cells synthesize IGFs and their six IGF binding proteins (IGFBP). IGFBP-5 was reported to stimulate bone cell growth in vitro and selected parameters of osteoblastic function in vivo, but its actual effects on bone formation are not established. We investigated the direct effects of IGFBP-5 on bone remodeling in two lines of transgenic mice overexpressing IGFBP-5 under the control of the osteocalcin promoter. Static and dynamic histomorphometry revealed that IGFBP-5 transgenic mice had a transient decrease in trabecular bone volume secondary to reduced trabecular number and thickness and a transient decrease in bone mineral apposition rate. Osteoblast number was normal, indicating impaired osteoblastic function. Osteoclast number and bone resorption were normal. Total, vertebral, and femoral bone mineral densities were reduced in IGFBP-5 transgenics by 14-27% at 4 wk of age, but not in older animals. Stromal cells expressing the IGFBP-5 transgene displayed decreased expression of alkaline phosphatase, osteocalcin, core binding factor 1, and type I collagen transcripts when compared with cells from wild-type animals. In conclusion, transgenic mice overexpressing IGFBP-5 in the bone microenvironment have a transient decrease in trabecular bone volume, impaired osteoblastic function, and osteopenia.

Alkaline Phosphatase↗
PubMed2001

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.

Animals↗
PubMed2001

The metastasis-associated metalloproteinase stromelysin-3 is induced by transforming growth factor-beta in osteoblasts and fibroblasts.

Bone matrix serves as a reservoir of growth factors important in growth and tissue remodeling, and transforming growth factor-beta (TGF-beta) is abundant in bone matrix. Normal processes, such as remodeling, and pathological processes, such as osteolytic metastasis, cause the release of growth factors from the matrix, allowing them to influence the behavior of cells within their microenvironment. Breast cancer metastases frequently establish themselves in the bone compartment, often causing localized osteolysis. Stromelysin-3 is a matrix metalloproteinase associated with tumor metastases. Its expression in host tissues favors the homing and survival of malignant epithelial cells in early tumorigenesis by releasing and/or activating growth factors sequestered in the extracellular matrix. Osteoblasts express stromelysin-3, and Northern and Western blot analysis show that its messenger RNA and protein levels are increased by TGF-beta. Nuclear run-off assays demonstrate activation of gene transcription, and experiments using transcription inhibitors demonstrate stabilization of stromelysin-3 messenger RNA by TGF-beta. Importantly, TGFbeta induces stromelysin-3 in fibroblasts by similar mechanisms, indicating that it is likely to stimulate stromelysin-3 expression in breast stroma. Stimulation of stromelysin-3 expression by TGF-beta in fibroblasts and osteoblasts could play a role in the metastasis of breast cancer cells and their homing and survival in bone.

Animals↗
PubMed2001

Glucocorticoid suppression of IGF I transcription in osteoblasts.

Glucocorticoids have profound effects on bone formation, decreasing IGF I transcription in osteoblasts, but the mechanisms involved are poorly understood. We previously showed that the bp +34 to +192 region of the rat IGF I exon 1 promoter was responsible for repression of IGF I transcription by cortisol in cultures of osteoblasts from fetal rat calvariae (Ob cells). Here, site-directed mutagenesis was used to show that a binding site for members of the CAAT/enhancer binding protein family of transcription factors, within the +132 to +158 region of the promoter, mediates this glucocorticoid effect. EMSAs demonstrated that cortisol increased binding of osteoblast nuclear proteins to the +132 to +158 region of the IGF I promoter. Supershift assays showed that CAAT/enhancer binding protein alpha, beta, and delta interact with this sequence, and binding of CAAT/enhancer binding protein delta, in particular, was increased in the presence of cortisol. Northern blot analysis showed that CAAT/enhancer binding protein delta and beta transcripts were increased by cortisol in Ob cells. Further, cortisol increased the transcription of these genes and increased the stability of CAAT/enhancer binding protein delta mRNA. In conclusion, cortisol represses IGF I transcription in osteoblasts, and CAAT/enhancer binding proteins appear to play a role in this effect.

Animals↗
PubMed2000

Bone morphogenetic protein-4 regulates its own expression in cultured osteoblasts.

During development, bone morphogenetic proteins (BMPs) induce the differentiation of mesenchymal progenitor cells to enter into the osteoblastic lineage, and BMPs enhance osteoblastic function. BMPs and noggin, a specific binding protein that blocks BMP actions, are expressed by osteoblastic cells but there is limited information about regulation of BMP synthesis in skeletal cells. We tested for the expression and regulation of BMP-4 in cultures of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells). BMP-4 caused a short-lived increase in BMP-4 mRNA followed by a marked inhibition of BMP-4 expression. The stimulatory effect was transcriptional, as determined by nuclear run-on assays, whereas the inhibitory effect was transcriptional and posttranscriptional, because longer BMP-4 exposure decreased its rate of transcription and shortened the half-life of BMP-4 mRNA in transcriptionally arrested Ob cells. BMP-2 and BMP-6 also inhibited BMP-4 mRNA levels. Transforming growth factor beta1 increased, whereas fibroblast growth factor-2, platelet-derived growth factor BB, and insulin-like growth factor I decreased BMP-4 mRNA in Ob cells. BMP-2 also was expressed by Ob cells and it was downregulated by BMP-2, BMP-4, and BMP-6. Noggin increased BMP-4 transcripts, suggesting autocrine control of BMP-4 expression. In conclusion, BMP-4 inhibits its own expression in Ob cells, a mechanism to limit BMP availability to osteoblasts.

Animals↗
PubMed2000

Platelet-derived growth factor induces collagenase 3 transcription in osteoblasts through the activator protein 1 complex.

Platelet-derived growth factor (PDGF) BB is a mitogen that stimulates bone resorption and increases collagenase 3 transcription in osteoblasts, although the mechanisms involved are as yet unknown. We examined the effect of PDGF BB on collagenase 3 transcription in cultures of osteoblasts from fetal rat calvariae (Ob cells). PDGF BB increased the activity of collagenase 3 promoter fragments transiently transfected into Ob cells. Deletion analysis of the collagenase promoter revealed three regions that impaired the induction of collagenase 3 by PDGF BB. A construct spanning base pair -53 to +28 collagenase 3 sequences, in relation to the start site of transcription +1, was fully responsive to PDGF BB and was studied in detail. Targeted mutations of an AP-1 site in this fragment decreased basal collagenase promoter activity and the responsiveness to PDGF BB, whereas mutations of Stat3 and Ets binding sites did not alter the response to PDGF. Electrophoretic mobility shift assay, using nuclear extracts from control and treated cells, revealed AP-1 nuclear protein complexes that were enhanced in extracts from PDGF BB-treated Ob cells. Supershift assays revealed that antibodies to c-Fos, Fos B, Fra-2, c-Jun, Jun B, and Jun D shifted the binding of nuclear extracts from cells treated with PDGF BB to AP-1 sequences. In conclusion, PDGF BB induces collagenase 3 transcription in osteoblasts by regulating nuclear proteins interacting with AP-1 sequences.

Animals↗
PubMed2000

Transforming growth factor-beta increases interleukin-6 transcripts in osteoblasts.

Bone remodeling is regulated by local factors and cytokines. Among them, interleukin-6 (IL-6) plays a critical role in bone resorption, and its synthesis is stimulated by osteoresorptive factors. Transforming growth factor-beta (TGF-beta) is present in high amounts in the bone matrix and is a local regulator of bone formation. However, its role in bone resorption remains unclear. In this paper, we report that TGF-beta stimulates IL-6 transcripts in a time- and dose-dependent manner in primary rat osteoblasts isolated from 22-day-old calvariae (Ob cells). The TGF-beta effect on IL-6 mRNA levels does not require de novo protein synthesis because cycloheximide, a protein synthesis inhibitor, does not block the induction. The mechanisms of IL-6 stimulation by TGF-beta is at least partially transcriptional because TGF-beta induces IL-6 heterogenous nuclear RNA, and, to a lesser extent, IL-6 transcription rate as determined by a nuclear run-on assay. Transforming growth factor-beta upregulation of IL-6 may be critical in conditions of increased bone resorption, such as myeloma.

Animals↗
PubMed2000

Cortisol inhibits hepatocyte growth factor/scatter factor expression and induces c-met transcripts in osteoblasts.

Hepatocyte growth factor/scatter factor (HGF/SF) is expressed by osteoblasts and has important effects on repair and bone remodeling. Because glucocorticoids regulate these two functions, we tested the effects of cortisol on the expression of HGF/SF and c-met, the protooncogene encoding the HGF/SF receptor, in cultures of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells). Cortisol decreased HGF/SF mRNA levels and diminished the induction of HGF/SF transcripts by fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor BB (PDGF BB). Cortisol also decreased FGF-2 and PDGF BB-induced HGF/SF mRNA and polypeptide levels in MC3T3 cells. In contrast, cortisol enhanced the expression of c-met transcripts in Ob cells. Cortisol did not modify the half-life of HGF/SF or of c-met mRNA in transcriptionally arrested cells, and it increased the rate of transcription of c-met. In conclusion, cortisol decreases HGF/SF transcripts in Ob cells and enhances c-met expression transcriptionally. The effects of cortisol on HGF/SF could be relevant to its inhibitory actions on bone formation and repair.

Animals↗