Biomedical subjects

D Durant

Publications and source records attributed to D Durant.

13 recordsLinked to original sources

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↗

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

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

Transcriptional regulation of connective tissue growth factor by cortisol in osteoblasts.

Glucocorticoids have important effects on osteoblastic function. Connective tissue growth factor (CTGF)/insulin-like growth factor binding protein-related protein 2 (IGFBP-rP2) plays a role in cell adhesion and function. We examined the regulation of CTGF/IGFBP-rP2 synthesis in cultures of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells). Cortisol caused a time- and dose-dependent increase in CTGF/IGFBP-rP2 mRNA levels in Ob cells. Cycloheximide did not preclude the effect, indicating that it was not protein synthesis dependent. Cortisol increased the rate of CTGF/IGFBP-rP2 transcription and, in transcriptionally arrested Ob cells, did not modify the decay of the transcript. Parathyroid hormone decreased, whereas transforming growth factor-beta and, to a lesser extent, bone morphogenetic protein 2 increased CTGF/IGFBP-rP2 mRNA levels, but other hormones and growth factors had no effect. In conclusion, cortisol stimulates CTGF/IGFBP-rP2 transcription in Ob cells. Because CTGF/IGFBP-rP2 binds IGFs, its increased expression could be relevant to the actions of cortisol in bone.

Animals↗

PubMed1999

Platelet-derived growth factor induces interleukin-6 transcription in osteoblasts through the activator protein-1 complex and activating transcription factor-2.

Platelet-derived growth factor (PDGF) BB, a mitogen that stimulates bone resorption, increases the expression of interleukin-6 (IL-6), a cytokine that induces osteoclast recruitment. The mechanisms involved in IL-6 induction by PDGF BB are poorly understood. We examined the effect of PDGF BB on IL-6 expression in cultures of osteoblasts from fetal rat calvariae (Ob cells). PDGF BB increased IL-6 mRNA and heterogeneous nuclear RNA levels, the rate of transcription, and the activity of base pairs (bp) -2906 to +20 IL-6 promoter fragments transiently transfected into Ob cells. Deletion analysis revealed two responsive regions, one containing an activator protein-1 (AP-1) site located between bp -276 and -257, and a second, less well defined, downstream of -257. Targeted mutations of a cyclic AMP-responsive element (CRE), and nuclear factor-IL-6 and nuclear factor-kappaB binding sites in a bp -257 to +20 IL-6 construct that was transfected into Ob cells, revealed that the CRE also contributed to IL-6 promoter induction by PDGF BB. Electrophoretic mobility shift assay revealed AP-1 and CRE nuclear protein complexes that were enhanced by PDGF BB. Supershift assays revealed binding of Jun and Fos to AP-1 and CRE sequences and binding of activating transcription factor-2 to CRE. In conclusion, PDGF BB induces IL-6 transcription in osteoblasts by regulating nuclear proteins of the AP-1 complex and activating transcription factor-2.

Activating Transcription Factor 2↗

PubMed1997

Interleukin-6 with its soluble receptor enhances the expression of insulin-like growth factor-I in osteoblasts.

Interleukin (IL)-6, a cytokine produced by skeletal cells and known to increase bone resorption, has mitogenic effects for bone cells, possibly by regulating the synthesis of other local factors. We tested the effects of IL-6 and its soluble receptor (IL-6sR) on the expression of insulin-like growth factor (IGF)-I and IGF-II in cultured osteoblast-enriched cells from fetal rat calvariae (Ob cells). IL-6 did not modify IGF-I messenger RNA (mRNA) levels, but when tested in the presence of IL-6sR, IL-6 at 1 to 100 ng/ml increased IGF-I transcripts by up to 3.2-fold after 24 h. IL-6sR caused a small increase in IGF-I mRNA levels when tested alone. IL-6 and IL-6sR increased immunoreactive IGF-I levels by 2.4-fold after 24 h and 6.4-fold after 48 h. Cycloheximide prevented, and indomethacin markedly decreased, the effect of IL-6 and IL-6sR on IGF-I mRNA levels, but hydroxyurea did not. IL-6 and IL-6sR did not alter the decay of IGF-I mRNA in transcriptionally arrested Ob cells, and the half-life of the predominant 6.5-kb IGF-I transcript was about 11 h in control and treated cells. In addition, IL-6 and IL-6sR increased the levels of IGF-I heterogeneous nuclear RNA. IL-11 also increased IGF-I mRNA levels, whereas oncostatin M and leukemia-inhibitory factor did not. In contrast to their effects on IGF-I, IL-6 and IL-6sR caused only a modest increase in IGF-II mRNA and polypeptide levels. In conclusion, IL-6, in the presence of IL-6sR, increases IGF-I synthesis in Ob cells; this effect may lead to a secondary increase in bone formation.

Animals↗

PubMed1997

Interleukin-6 and its soluble receptor regulate the expression of insulin-like growth factor binding protein-5 in osteoblast cultures.

Interleukin-6 (IL-6), a cytokine produced by bone cells, is known to influence bone resorption by stimulating the development of osteoclasts from precursor cells and to have mitogenic actions on osteoblastic cells. Insulin-like growth factors (IGFs) are important local regulators of bone formation, and IGF binding protein (IGFBP)-5 stimulates bone cell growth and enhances the effects of IGF-I. We tested the effects of IL-6 in the presence and absence of its soluble receptor (sIL-6R) on IGFBP-5 expression in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). When tested individually, IL-6 and sIL-6R had a modest stimulatory effect on IGFBP-5 messenger RNA (mRNA) levels. In contrast, when IL-6 and sIL-6R were tested in combination, they caused a considerable increase in IGFBP-5 mRNA levels, and IL-6 at 100 ng/ml and sIL-6R at 125 ng/ml increased IGFBP-5 transcripts by 5- to 7-fold after 24 h. The effect of IL-6 and sIL-6R on IGFBP-5 transcripts was not blocked by indomethacin, but cycloheximide markedly inhibited IGFBP-5 mRNA levels in control and treated cultures. IL-6 and sIL-6R did not modify the decay of IGFBP-5 mRNA in transcriptionally arrested Ob cells, and stimulated the rate of IGFBP-5 transcription as demonstrated by a nuclear run-on assay. IL-6 and sIL-6R did not increase intact IGFBP-5 levels in the extracellular matrix and increased IGFBP-5 fragments in the culture medium. Conditioned medium from Ob cells induced the proteolytic fragmentation of an IGFBP-5 standard, an effect that was accelerated and enhanced by conditioned medium from IL-6/sIL-6R-treated cultures and prevented by metalloprotease inhibitors. In conclusion, IL-6, in the presence of sIL-6R, stimulates IGFBP-5 mRNA expression in Ob cells by transcriptional mechanisms, and accelerates the fragmentation of the protein.

Animals↗

PubMed1988