Cultures Of Osteoblast-enriched Cells Research Articles

FGF Suppresses Poldip2 Expression in Osteoblasts.

Osteoporosis is one of the most prevalent ageing-associated diseases that are soaring in the modern world. Although various aspects of the disease have been investigated to understand the bases of osteoporosis, the pathophysiological mechanisms underlying bone loss is still incompletely understood. Poldip2 is a molecule that has been shown to be involved in cell migration of vascular cells and angiogenesis. However, expression of Poldip2 and its regulation in bone cells were not known. Therefore, we examined the Poldip2 mRNA expression and the effects of bone regulators on the Poldip2 expression in osteoblasts. We found that Poldip2 mRNA is expressed in osteoblastic MC3T3-E1 cells. As FGF controls osteoblasts and angiogenesis, FGF regulation was investigated in these cells. FGF suppressed the expression of Poldip2 in MC3T3-E1 cells in a time dependent manner. Protein synthesis inhibitor but not transcription inhibitor reduced the FGF effects on Poldip2 gene expression in MC3T3-E1 cells. As for bone-related hormones, dexamethasone was found to enhance the expression of Poldip2 in osteoblastic MC3T3-E1 cells whereas FGF still suppressed such dexamethasone effects. With respect to function, knockdown of Poldip2 by siRNA suppressed the migration of MC3T3-E1 cells. Poldip2 was also expressed in the primary cultures of osteoblast-enriched cells and FGF also suppressed its expression. Finally, Poldip2 was expressed in femoral bone in vivo and its levels were increased in aged mice compared to young adult mice. These data indicate that Poldip2 is expressed in osteoblastic cells and is one of the targets of FGF. J. Cell. Biochem. 118: 1670-1677, 2017. © 2016 Wiley Periodicals, Inc.

Regional Dura Mater Differentially Regulates Osteoblast Gene Expression

Recent studies have suggested that regionally differentiated dura mater regulates murine cranial suture fate by providing growth factors to the osteoblasts in the overlying suture complex. To determine if regionally differentiated dura mater is capable of effecting changes in osteoblast gene expression, an in vitro coculture system was established in which osteoblast-enriched cell cultures derived from neonatal rat calvaria were grown in serum-free media in the presence of dural cells derived from posterior frontal (PF) or sagittal (SAG) dural tissues, recapitulating the in situ relation between the underlying dura mater and the osteoblasts in the overlying cranial suture. In this study, the changes in osteoblast gene expression induced by signaling from regional dura mater were examined by analyzing total cellular RNA isolated from osteoblasts cocultured with PF or SAG dural cells. The expression of extracellular matrix molecules (alkaline phosphatase, bone sialoprotein, osteopontin, and osteocalcin) and the transcription factor Msx2 was assessed. Consistent with previous data, the findings demonstrate that osteoblasts cocultured with dural cells undergo changes in gene expression indicative of a more differentiated osteoblast. Additionally, the data suggest that regionally differentiated dura mater isolated from the PF suture enhances the expression of osteogenic genes to a greater extent than SAG suture-derived dural cells. These data support an osteoinductive role for suture-derived dural cells in vitro that may have implications for suture biology in vivo.

Bone Morphogenetic Proteins Induce Gremlin, a Protein That Limits Their Activity in Osteoblasts

Bone morphogenetic proteins (BMP) induce the differentiation of cells of the osteoblastic lineage and enhance the function of the osteoblast. Growth factor activity is regulated by binding proteins, and we previously showed that BMPs induce noggin, a glycoprotein that binds and blocks BMP action. Recently, additional BMP antagonists, such as gremlin, have been described, but there is no information about their expression or function in osteoblasts. We tested for the expression of gremlin and studied its induction by BMPs in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). BMP-2 caused a time- and dose-dependent increase in gremlin messenger RNA and polypeptide levels, as determined by Northern and Western blot analyses. The effects of BMP-2 on gremlin transcripts were independent of new protein synthesis. BMP-2 increased the rate of gremlin transcription as determined by nuclear run-on assays. Fibroblast growth factor-2 and platelet-derived growth factor BB also induced gremlin, but other hormones and growth factors had no effect. Gremlin prevented the stimulatory effects of BMP-2 on DNA, collagen, noncollagen protein synthesis, and alkaline phosphatase activity in Ob cells. In conclusion, BMPs induce gremlin transcription in Ob cells, a mechanism that probably limits BMP action in osteoblasts.

Bone morphogenetic proteins induce gremlin, a protein that limits their activity in osteoblasts.

Bone morphogenetic proteins (BMP) induce the differentiation of cells of the osteoblastic lineage and enhance the function of the osteoblast. Growth factor activity is regulated by binding proteins, and we previously showed that BMPs induce noggin, a glycoprotein that binds and blocks BMP action. Recently, additional BMP antagonists, such as gremlin, have been described, but there is no information about their expression or function in osteoblasts. We tested for the expression of gremlin and studied its induction by BMPs in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). BMP-2 caused a time- and dose-dependent increase in gremlin messenger RNA and polypeptide levels, as determined by Northern and Western blot analyses. The effects of BMP-2 on gremlin transcripts were independent of new protein synthesis. BMP-2 increased the rate of gremlin transcription as determined by nuclear run-on assays. Fibroblast growth factor-2 and platelet-derived growth factor BB also induced gremlin, but other hormones and growth factors had no effect. Gremlin prevented the stimulatory effects of BMP-2 on DNA, collagen, noncollagen protein synthesis, and alkaline phosphatase activity in Ob cells. In conclusion, BMPs induce gremlin transcription in Ob cells, a mechanism that probably limits BMP action in osteoblasts.

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.

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.

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.

Parathyroid hormone increases mac25/insulin-like growth factor-binding protein-related protein-1 expression in cultured osteoblasts.

PTH induces the synthesis of insulin-like growth factor I (IGF-I) and regulates the expression of IGF-binding proteins (IGFBP) in osteoblast cultures. IGFBP-related protein-1 (IGFBP-RP-1), the product of the mac25 gene, binds IGF-I, IGF-II, and insulin. We tested the actions of PTH on the expression of mac25/IGFBP-RP-1 in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). PTH at 0.1-10 nM for 6-48 h increased mac25/IGFBP-RP-1 messenger RNA (mRNA) levels in Ob cells, an effect not altered by cycloheximide. PGE2 increased mac25/IGFBP-RP-1 mRNA levels, but indomethacin did not modify basal or PTH-stimulated mac25/IGFBP-RP-1 expression. The decay of mac25/IGFBP-RP-1 mRNA in transcriptionally arrested Ob cells was not modified by PTH, and PTH increased the rate of IGFBP-RP-1 transcription. GH, insulin, bone morphogenetic protein-2, fibroblast growth factor-2, platelet-derived growth factor BB, IGF-I, and IGF-II did not modify mac25/IGFBP-RP-1 expression, whereas transforming growth factor-beta1 was modestly stimulatory. In conclusion, PTH stimulates mac25/IGFBP-RP-1 transcription in osteoblasts, an effect that could be relevant to the actions of PTH in bone.

Cortisol enhances the expression of mac25/insulin-like growth factor-binding protein-related protein-1 in cultured osteoblasts.

Glucocorticoids inhibit the synthesis of insulin-like growth factor I (IGF-I) and regulate the expression of IGF-binding proteins (IGFBPs) in osteoblast cultures. IGFBP-related protein-1 (IGFBP-rP1), the product of the mac25 gene, binds IGF-I, IGF-II, and insulin, and we postulated that glucocorticoids regulate IGFBP-rP1 synthesis in osteoblasts. We tested the expression of mac25/IGFBP-rP1 in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). Cortisol treatment at 10 nM to 1 microM for 24-48 h caused a time- and dose-dependent increase in mac25/IGFBP-rP1 messenger RNA (mRNA) levels in Ob cells. Cycloheximide at 3.6 microM did not alter mac25/IGFBP-rP1 transcripts in control or cortisol-treated cells. Cortisol did not modify the decay of mac25/IGFBP-rP1 mRNA in transcriptionally arrested Ob cells and increased the rate of IGFBP-rP1 transcription as determined by nuclear run-on assays. Retinoic acid also increased mac25/IGFBP-rP1 mRNA levels, but 17beta-estradiol, testosterone, 5alpha-dihydrotestosterone, progesterone, and 1,25-dihydroxyvitamin D3 did not. In conclusion, cortisol stimulates mac25/IGFBP-rP1 expression in Ob cells by transcriptional mechanisms. As IGFBP-rP1 binds and possibly modifies the effects of IGFs and insulin, its increased expression could be relevant to the inhibitory actions of cortisol in bone.

Bone morphogenetic proteins induce the expression of noggin, which limits their activity in cultured rat osteoblasts.

Bone morphogenetic proteins (BMPs) induce the differentiation of cells of the osteoblastic lineage and enhance the function of the osteoblast. Growth factors are regulated by binding proteins, but there is no information about binding proteins for BMPs in skeletal cells. Noggin specifically binds BMPs, but its expression by cells of the osteoblastic lineage has not been reported. We tested for the expression of noggin and its induction by BMP-2 in cultures of osteoblast-enriched cells from 22-d-old fetal rat calvariae (Ob cells). BMP-2 caused a time- and dose-dependent increase in noggin mRNA and polypeptide levels, as determined by Northern and Western blot analyses. The effects of BMP-2 on noggin transcripts were dependent on protein, but independent of DNA synthesis. BMP-2 increased the rates of noggin transcription as determined by nuclear run-on assays. BMP-4, BMP-6, and TGF-beta1 increased noggin mRNA in Ob cells, but basic fibroblast growth factor, platelet- derived growth factor BB, and IGF-I did not. Noggin decreased the stimulatory effects of BMPs on DNA and collagen synthesis and alkaline phosphatase activity in Ob cells. In conclusion, BMPs induce noggin transcription in Ob cells, a probable mechanism to limit BMP action in osteoblasts.

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 factor I inhibits the transcription of collagenase 3 in osteoblast cultures

Insulin-like growth factor (IGF) I is an autocrine regulator of bone remodeling which inhibits bone collagen degradation and interstitial collagenase 3 mRNA levels. The mechanism of this inhibitory effect on collagenase 3 expression is not known. We tested the effects of IGF I on collagenase 3 gene expression in cultures of osteoblast-enriched cells from 22 day fetal rat calvariae (Ob cells) to determine whether transcriptional or posttranscriptional mechanisms were involved in the regulation of the collagenase 3 gene. IGF I at 10-100 nM caused a dose-dependent decrease in collagenase mRNA and protein levels. IGF I did not modify the half-life of collagenase 3 mRNA in transcriptionally arrested Ob cells, whereas it decreased the levels of interstitial collagenase 3 heterogeneous nuclear RNA. In addition, IGF I decreased the rates of transcription of the collagenase gene and the activity of a 2.1 kilobase collagenase 3 promoter construct transiently transfected into Ob cells. In conclusion, IGF I decreases the expression of collagenase 3 mRNA by transcriptional mechanisms.

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 action 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-transretinoic 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, possible 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.

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.

Growth factor regulation of insulin-like growth factor binding protein-6 expression in osteoblasts.

Previously we have shown that transforming growth factor beta (TGF beta) 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 beta 1, 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 beta 1 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 beta 1 concentrations of 0.04 nM and higher. TGF beta 1 also decreased IGFBP-6 polypeptide levels in the medium, as determined by Western immunoblot analysis. Cycloheximide at 3.6 microM decreased IGFBP-6 transcripts and prevented the effect of TGF beta 1. The decay of IGFBP-6 mRNA in transcriptionally arrested Ob cells was not modified by TGF beta 1. In addition, TGF beta 1 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 beta 1 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 beta 1 could be a local feedback mechanism to increase the amount of IGF II available in the bone microenvironment.

Transforming growth factor beta1 inhibits collagenase 3 expression by transcriptional and post-transcriptional mechanisms in osteoblast cultures.

Transforming growth factor (TGF) beta1 is an autocrine regulator of bone cell function. We demonstrated that TGF beta1 enhances bone collagen synthesis, but its effects on collagen degradation are not well characterized. We tested the effects of TGF beta1 on rat collagenase 3 expression in cultures of osteoblast-enriched cells from fetal rat calvariae (Ob cells). Treatment with TGF beta1 at 0.4 nM decreased steady state collagenase mRNA levels after 2 to 24 h. This dose-dependent effect was observed at TGF beta1 concentrations of 4 pM to 1.2 nM, and was accompanied by decreased levels of immunoreactive procollagenase. The protein synthesis inhibitor cycloheximide increased collagenase transcripts, but did not prevent the effect of TGF beta1 on collagenase mRNA levels. TGF beta1 accelerated the decay of collagenase mRNA in transcriptionally arrested Ob cells. In addition, TGF beta1 decreased the levels of collagenase heterogeneous nuclear RNA and the rate of collagenase gene transcription in Ob cells. TGF beta1 enhanced the expression of tissue inhibitors of metalloproteinases (TIMP) 1 and 3 and caused a modest decrease of TIMP 2 mRNA levels. In conclusion, TGF beta1 decreases interstitial collagenase transcripts and protease levels in Ob cells by transcriptional and post-transcriptional mechanisms, and this effect may contribute to its actions on bone matrix.

Effect of (poly)-L-lactic acid on the proliferation and differentiation of primary bone cells in vitro.

A previous study has shown bone formation around poly-L-lactic acid (PLLA) wire in vivo. However, it is still unknown how bone cells are stimulated to form bone around PLLA wire. The effect of PLLA on primary bone cells in vitro is the subject of this study. Osteoprogenitor and osteoblastic cells derived from neonatal mouse calvaria were cultured after addition of PLLA wire or L-lactide to the culture medium. Alkaline phosphatase (AP) activity, as a parameter of bone cell differentiation, and DNA content, to assess cell growth were measured. In osteoblast-enriched cell cultures PLLA wire did not affect DNA content, but AP activity was increased by 28%. In osteoprogenitor-enriched cell cultures PLLA wire decreased DNA content by 10%, but AP activity of the cells was not affected. L-Lactide enhanced the DNA content of osteoblastic cell cultures at 0.1 mM by 19%, but not at higher concentrations, and did not affect cell differentiation. In osteoprogenitor cell cultures, L-lactide had no effect at all. These results indicate that the proliferation and differentiation of bone cells in vitro can be modulated by PLLA, dependent on their stage of differentiation. L-Lactide at concentrations up to 10 mM did not adversely affect cell growth or differentiation, while PLLA wire upregulated the expression of the osteoblastic phenotype. These data support the observation of newly formed bone around poly-lactic acid wire in vivo in other the studies.

Autocrine down-regulation of collagenase-3 in rat bone cell cultures by insulin-like growth factors

Insulin-like growth factors (IGF)-I and -II are presumed to act as autocrine regulators of bone formation. Recently, we demonstrated that IGF-I and -II inhibit bone collagen degradation and collagenase-3 synthesis in osteoblast cultures. Therefore, we tested the autocrine role of IGFs in the endogenous expression of collagenase-3 in cultures of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells). Steady-state messenger RNA (mRNA) levels were determined by Northern blot analysis and collagenase concentrations in the culture medium were determined by Western immunoblot. Basal level collagenase-3 transcripts decreased in Ob cell cultures, coinciding with an increase in IGF-I and -II protein levels. Removal of the conditioned medium modestly increased collagenase-3 mRNA levels and restored the ability of exogenously added IGF-I to repress collagenase-3 transcripts. IGF neutralizing antibodies and IGF binding proteins-2 and -3 in excess increased and sustained collagenase mRNA, heterogeneous nuclear RNA, and protease levels in Ob cell cultures. In conclusion, IGF-I and -II are autocrine repressors of collagenase-3 synthesis, and this effect may contribute to their actions on the maintenance of a normal bone collagen matrix.

Autocrine down-regulation of collagenase-3 in rat bone cell cultures by insulin-like growth factors.

Insulin-like growth factors (IGF)-I and -II are presumed to act as autocrine regulators of bone formation. Recently, we demonstrated that IGF-I and -II inhibit bone collagen degradation and collagenase-3 synthesis in osteoblast cultures. Therefore, we tested the autocrine role of IGFs in the endogenous expression of collagenase-3 in cultures of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells). Steady-state messenger RNA (mRNA) levels were determined by Northern blot analysis and collagenase concentrations in the culture medium were determined by Western immunoblot. Basal level collagenase-3 transcripts decreased in Ob cell cultures, coinciding with an increase in IGF-I and -II protein levels. Removal of the conditioned medium modestly increased collagenase-3 mRNA levels and restored the ability of exogenously added IGF-I to repress collagenase-3 transcripts. IGF neutralizing antibodies and IGF binding proteins-2 and -3 in excess increased and sustained collagenase mRNA, heterogeneous nuclear RNA, and protease levels in Ob cell cultures. In conclusion, IGF-I and -II are autocrine repressors of collagenase-3 synthesis, and this effect may contribute to their actions on the maintenance of a normal bone collagen matrix.

Expression and growth factor regulation of platelet-derived growth factor B transcripts in primary osteoblast cell cultures.

Platelet-derived growth factor (PDGF), an important bone cell mitogen, exists as a homo- or heterodimer product of the PDGF-A and -B genes. Normal unstimulated cells of the osteoblast lineage express the PDGF-A gene, but it is not known whether they express the PDGF-B gene. We examined the expression of PDGF-B messenger RNA (mRNA) levels in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells) and determined whether they were modified by transforming growth factor-beta 1 (TGF beta 1), basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I), and PDGF-BB. Ob cells expressed PDGF-B transcripts of 3.5 kilo-bases, as determined by Northern blot analysis. Treatment of Ob cells with TGF beta 1 at 0.01-1.2 nM caused a dose-dependent increase in steady state PDGF-B mRNA, an effect that was initially observed after 2 h and was maximal after 6h. Cycloheximide induced PDGF-B transcripts and decreased the effect of TGF beta 1. TGF beta 1 did not modify the half-life of PDGF-B mRNA in transcriptionally arrested Ob cells and increased the rate of PDGF-B gene transcription in nuclear run-on assays. In contrast, treatment with PDGF-BB at 3.3 nM, bFGF at 6 nM, or IGF-I at 100 nM for 2-24 h did not modify PDGF-B mRNA levels in Ob cells. In conclusion, normal Ob cells express the PDGF-B gene, and TGF beta 1 induces its transcription, whereas bFGF, IGF-I, and PDGF-BB do not enhance the levels of PDGF-B mRNA. PDGF-BB may act not only as a systemic but also as a local regulator of bone cell function.