Bari Gabbitas

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.

Retinoic acid stimulates the transcription of insulin-like growth factor binding protein-6 in skeletal cells

Retinoic acid has important actions on cell differentiation and osteoblastic function, and some of these actions may be mediated by changes in the insulin‐like growth factor (IGF) axis. Skeletal cells synthesize IGF I and II and the six known IGF binding proteins (IGFBP). IGFBP‐6 binds IGF II with high affinity and prevents IGF II‐mediated effects. In fibroblasts, IGFBP‐6 levels are regulated by retinoic acid, and we postulated that retinoic acid may regulate IGF II in bone by altering IGFBP‐6 synthesis. We examined the effect of retinoic acid on IGFBP‐6 expression in cultures of osteoblast‐enriched cells from 22‐day fetal rat calvariae (Ob cells). Retinoic acid caused a time‐ and dose‐dependent increase in IGFBP‐6 mRNA levels, as determined by Northern blot analysis. The effect was maximal after 48 h of treatment and observed with retinoic acid at concentrations of 10 nM to 1 μM. Retinoic acid increased IGFBP‐6 polypeptide levels in the culture medium, as determined by Western immunoblot analysis. Cycloheximide at 3.6 μM slightly decreased IGFBP‐6 transcripts but did not prevent the stimulatory effect of retinoic acid. The decay of IGFBP‐6 mRNA in transcriptionally arrested Ob cells was similar in control and retinoic acid‐treated cells, and retinoic acid increased the rates of IGFBP‐6 transcription, as determined by nuclear run on assays. In conclusion, retinoic acid enhances IGFBP‐6 expression in Ob cells by transcriptional mechanisms. Since IGFBP‐6 prevents the effects of IGF II, increased synthesis of IGFBP‐6 could mediate selected actions of retinoic acid in bone.

Retinoic acid regulates the expression of insulin-like growth factors I and II in osteoblasts

Insulin‐like growth factors (IGF) I and II are the most abundant growth factors secreted by skeletal cells, and retinoic acid has many important actions on cell differentiation and osteoblastic function. Some of these actions may be mediated by changes in the expression of IGF I and II since IGFs are known to enhance the differentiated function of the osteoblast. We examined the effects of all‐trans‐retinoic acid on IGF I and IGF II expression in cultures of osteoblast‐enriched cells from 22 day fetal rat calvariae (Ob cells). Retinoic acid caused a transient increase in IGF I and IGF II mRNA levels after 6 h, but after 24 and 48 h of treatment a dose‐dependent decrease was observed. Cycloheximide prevented the inhibitory effect of retinoic acid. Retinoic acid treatment for 48 h decreased IGF I polypeptide levels in the culture medium. In contrast, 48 h exposure to retinoic acid increased IGF II polypeptide levels, possibly due to increased levels of IGF binding protein‐6. The decay of IGF I and II mRNA in transcriptionally arrested Ob cells was similar in control and retinoic acid‐treated cells. After 2 h, retinoic acid increased the rates of IGF I and II transcription, as determined by a nuclear run‐on assay and heterogeneous nuclear RNA levels, but after 24 h retinoic acid was inhibitory. Retinoic acid had opposite effects to IGFs in osteoblasts and inhibited DNA and collagen synthesis. In conclusion, following a small transient increase, retinoic acid causes a pronounced decrease in IGF I and IGF II mRNA expression in Ob cells. However, treatment with retinoic acid causes a decrease in IGF I and an increase in IGF II polypeptide levels. These changes in the IGF/IGFBP axis may be relevant to the mechanism of action of retinoic acid in bone. J. Cell. Physiol. 172:253–264, 1997.

GROWTH FACTOR REGULATION OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-6 EXPRESSION IN OSTEOBLASTS

Previously we have shown that transforming growth factor β (TGF β) 1, basic fibroblast growth factor (FGF), and platelet‐derived growth factor (PDGF) BB inhibit the synthesis of insulin‐like growth factor (IGF) II, but their effects on IGF binding protein (IGFBP)‐6 in osteoblast cultures are not known. IGFBP‐6 binds IGF II with high affinity and prevents IGF II–mediated effects, so that a possible mode of regulating the IGF II available to bone cells would be by changing the levels of IGFBP‐6. To enhance our understanding of the actions of growth factors on the IGF II axis in bone, we tested the effects of TGF β1, basic FGF, PDGF BB, IGF I, and IGF II on the expression of IGFBP‐6 in cultures of osteoblast‐enriched cells from 22 day fetal rat calvariae (Ob cells). Treatment of Ob cells with TGF β1 caused a time‐ and dose‐dependent decrease in IGFBP‐6 mRNA levels, as determined by Northern blot analysis. The effect was maximal after 48 h and observed with TGF β1 concentrations of 0.04 nM and higher. TGF β1 also decreased IGFBP‐6 polypeptide levels in the medium, as determined by Western immunoblot analysis. Cycloheximide at 3.6 μM decreased IGFBP‐6 transcripts and prevented the effect of TGF β1. The decay of IGFBP‐6 mRNA in transcriptionally arrested Ob cells was not modified by TGF β1. In addition, TGF β1 decreased the rates of IGFBP‐6 transcription as determined by a nuclear run‐on assay. In contrast, basic FGF, PDGF BB, IGF I, and IGF II did not change IGFBP‐6 mRNA levels in Ob cells. In conclusion, TGF β1 inhibits IGFBP‐6 expression in Ob cells by transcriptional mechanisms. Since IGFBP‐6 binds IGF II and prevents its effects on bone cells, decreased synthesis of IGFBP‐6 induced by TGF β1 could be a local feedback mechanism to increase the amount of IGF II available in the bone microenvironment. J. Cell. Biochem. 66:77–86, 1997.

Insulin-like growth factors sustain insulin-like growth factor-binding protein-5 expression in osteoblasts

Insulin-like growth factors (IGFs) I and II are considered to be autocrine regulators of bone cell function. Recently, we demonstrated that IGF-I induces IGF-binding protein-5 (IGFBP-5) expression in cultures of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells). In the present study, we postulated that IGFs play an autocrine role in the maintenance of IGFBP-5 basal expression in Ob cells. IGFBP-2 and -3, at concentrations that bind endogenous IGFs, decreased IGFBP-5 mRNA levels, as determined by Northern blot analysis, and protein levels, as determined by Western immunoblots of extracellular matrix extracts of Ob cells. IGFBP-2 and -3 in excess inhibited IGFBP-5 heterogeneous nuclear RNA levels, as determined by RT-PCR, and did not alter the half-life of IGFBP-5 mRNA in transcriptionally arrested Ob cells. In conclusion, blocking endogenous IGFs in Ob cells represses IGFBP-5 expression, suggesting that IGFs are autocrine inducers of IGFBP-5 synthesis in osteoblasts.Insulin-like growth factors (IGFs) I and II are considered to be autocrine regulators of bone cell function. Recently, we demonstrated that IGF-I induces IGF-binding protein-5 (IGFBP-5) expression in cultures of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells). In the present study, we postulated that IGFs play an autocrine role in the maintenance of IGFBP-5 basal expression in Ob cells. IGFBP-2 and -3, at concentrations that bind endogenous IGFs, decreased IGFBP-5 mRNA levels, as determined by Northern blot analysis, and protein levels, as determined by Western immunoblots of extracellular matrix extracts of Ob cells. IGFBP-2 and -3 in excess inhibited IGFBP-5 heterogeneous nuclear RNA levels, as determined by RT-PCR, and did not alter the half-life of IGFBP-5 mRNA in transcriptionally arrested Ob cells. In conclusion, blocking endogenous IGFs in Ob cells represses IGFBP-5 expression, suggesting that IGFs are autocrine inducers of IGFBP-5 synthesis in osteoblasts.