Osteoblast-enriched Cultures Research Articles

Impairment of bone microstructure and upregulation of osteoclastogenic markers in spontaneously hypertensive rats

Hypertension and osteoporosis are the major non-communicable diseases in the elderly worldwide. Although clinical studies reported that hypertensive patients experienced significant bone loss and likelihood of fracture, the causal relationship between hypertension and osteoporosis has been elusive due to other confounding factors associated with these diseases. In this study, spontaneously hypertensive rats (SHR) were used to address this relationship and further explored the biophysical properties and the underlying mechanisms. Long bones of the hind limbs from 18-week-old female SHR were subjected to determination of bone mineral density (BMD) and their mechanical properties. Using synchrotron radiation X-ray tomographic microscopy (SRXTM), femoral heads of SHR displayed marked increase in porosity within trabecular area together with decrease in cortical thickness. The volumetric micro-computed tomography also demonstrated significant decreases in trabecular BMD, cortical thickness and total cross-sectional area of the long bones. These changes also led to susceptibility of the long bones to fracture indicated by marked decreases in yield load, stiffness and maximum load using three-point bending tests. At the cellular mechanism, an increase in the expression of osteoclastogenic markers with decrease in the expression of alkaline phosphatase was found in primary osteoblast-enriched cultures isolated from long bones of these SHR suggesting an imbalance in bone remodeling. Taken together, defective bone mass and strength in hypertensive rats were likely due to excessive bone resorption. Development of novel therapeutic interventions that concomitantly target hypertension and osteoporosis should be helpful in reduction of unwanted outcomes, such as bone fractures, in elderly patients.

Sustained Notch2 signaling in osteoblasts, but not in osteoclasts, is linked to osteopenia in a mouse model of Hajdu-Cheney syndrome

Individuals with Hajdu-Cheney syndrome (HCS) present with osteoporosis, and HCS is associated with NOTCH2 mutations causing deletions of the proline-, glutamic acid-, serine-, and threonine-rich (PEST) domain that are predicted to enhance NOTCH2 stability and cause gain-of-function. Previously, we demonstrated that mice harboring Notch2 mutations analogous to those in HCS (Notch2HCS) are severely osteopenic because of enhanced bone resorption. We attributed this phenotype to osteoclastic sensitization to the receptor activator of nuclear factor-κB ligand and increased osteoblastic tumor necrosis factor superfamily member 11 (Tnfsf11) expression. Here, to determine the individual contributions of osteoclasts and osteoblasts to HCS osteopenia, we created a conditional-by-inversion (Notch2COIN ) model in which Cre recombination generates a Notch2ΔPEST allele expressing a Notch2 mutant lacking the PEST domain. Germ line Notch2COIN inversion phenocopied the Notch2HCS mutant, validating the model. To activate Notch2 in osteoclasts or osteoblasts, Notch2COIN mice were bred with mice expressing Cre from the Lyz2 or the BGLAP promoter, respectively. These crosses created experimental mice harboring a Notch2ΔPEST allele in Cre-expressing cells and control littermates expressing a wild-type Notch2 transcript. Notch2COIN inversion in Lyz2-expressing cells had no skeletal consequences and did not affect the capacity of bone marrow macrophages to form osteoclasts in vitro In contrast, Notch2COIN inversion in osteoblasts led to generalized osteopenia associated with enhanced bone resorption in the cancellous bone compartment and with suppressed endocortical mineral apposition rate. Accordingly, Notch2 activation in osteoblast-enriched cultures from Notch2COIN mice induced Tnfsf11 expression. In conclusion, introduction of the HCS mutation in osteoblasts, but not in osteoclasts, causes osteopenia.

Activin A Suppresses Osteoblast Mineralization Capacity by Altering Extracellular Matrix (ECM) Composition and Impairing Matrix Vesicle (MV) Production

During bone formation, osteoblasts deposit an extracellular matrix (ECM) that is mineralized via a process involving production and secretion of highly specialized matrix vesicles (MVs). Activin A, a transforming growth factor-β (TGF-β) superfamily member, was previously shown to have inhibitory effects in human bone formation models through unclear mechanisms. We investigated these mechanisms elicited by activin A during in vitro osteogenic differentiation of human mesenchymal stem cells (hMSC). Activin A inhibition of ECM mineralization coincided with a strong decline in alkaline phosphatase (ALP(1)) activity in extracellular compartments, ECM and matrix vesicles. SILAC-based quantitative proteomics disclosed intricate protein composition alterations in the activin A ECM, including changed expression of collagen XII, osteonectin and several cytoskeleton-binding proteins. Moreover, in activin A osteoblasts matrix vesicle production was deficient containing very low expression of annexin proteins. ECM enhanced human mesenchymal stem cell osteogenic development and mineralization. This osteogenic enhancement was significantly decreased when human mesenchymal stem cells were cultured on ECM produced under activin A treatment. These findings demonstrate that activin A targets the ECM maturation phase of osteoblast differentiation resulting ultimately in the inhibition of mineralization. ECM proteins modulated by activin A are not only determinant for bone mineralization but also possess osteoinductive properties that are relevant for bone tissue regeneration.

Prostaglandin E2 Increases Transforming Growth Factor-β Type III Receptor Expression through CCAAT Enhancer-Binding Protein δ in Osteoblasts

Variations in individual TGF-beta receptors (TbetaRs) may modify TGF-beta activity and significantly alter its effects on connective tissue growth or repair. Differences in the amount of TbetaR type III (TbetaRIII) relative to signal transducing TbetaRI occur on bone cells during differentiation or in response to other growth regulators. Here we investigated prostaglandin (PG) E2, a potent effector during trauma, inflammation, or mechanical load, on TbetaR expression in primary osteoblast-enriched cultures. PGE2 rapidly increased TbetaRIII mRNA and protein expression and enhanced TbetaRIII gene promoter activity through a discrete region within 0.4 kb of the transcription start site. PGE2 alters osteoblast function through multiple signal-inducing pathways. In this regard, protein kinase A (PKA) activators, PGE1 and forskolin, also enhanced gene expression through the TbetaRIII gene promoter, whereas protein kinase C activators, PGF2alpha and phorbol myristate acetate, did not. The stimulatory effect of PGE2 on TbetaRIII promoter activity was suppressed by a dominant negative PKA-regulatory subunit, but not by dominant negative protein kinase C. PGE2 specifically increased nuclear factor CCAAT enhancer-binding protein delta (C/EBPdelta) binding to a half-binding site upstream of the basal TbetaRIII promoter region, and promoter activity was sensitive to C/EBPdelta overexpression and to dominant-negative C/EBPdelta competition. In parallel with their effect on TbetaRIII expression, activators of PKA decreased TGF-beta-induced activity. In summary, high levels of PGE2 that occur with inflammation or trauma may, through PKA-activated C/EBPdelta, preferentially increase TbetaRIII expression and in this way delay TGF-beta-dependent activation of osteoblasts during the early stabilization phase of bone repair.

Effects of Nonsteroidal Anti-Inflammatory Drugs on Transforming Growth Factor-β Expression and Bioactivity in Rat Osteoblast-Enriched Cultures

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been reported to suppress bone remodeling in normal and repaired bones. Our previous results indicated that ketorolac and indomethacin suppressed proliferation, stimulated early differentiation, and induced apoptosis in cultured osteoblasts. Transforming growth factor-beta (TGF-beta) has been reported to enhance proliferation, suppress differentiation, and prevent apoptosis in osteoblasts. We proposed that one pathway of NSAID effects on osteoblast function might be through inhibition of the expression and/or bioactivity of TGF-beta in osteoblasts. We tested the effects of ketorolac and indomethacin on the expression of TGF-beta1 mRNA and protein and the bioactivity of TGF-beta in osteoblast-enriched cultures derived from fetal calvaria. The effects of prostaglandin E1 (PGE1) and PGE2 on TGF-beta expression and bioactivity were also examined in order to understand more about the role of prostaglandins in osteoblast function. Simultaneously, we estimated whether these NSAID effects on osteoblasts were prostaglandin-related. The results showed that 24-hour treatments with both PGEs and theoretic therapeutic concentrations of ketorolac and indomethacin had no significant effects on the levels of either transcription or translation of TGF-beta or the post-translational function of TGF-beta in osteoblasts. These results suggest that NSAIDs do not affect osteoblast function through changes in TGF-beta action in osteoblasts.

Osteoblasts subjected to spaceflight and simulated space shuttle launch conditions.

To understand further the effects of spaceflight on osteoblast-enriched cultures, normal chicken calvarial osteoblasts were flown aboard shuttle flight STS-77, and the total number of attached cells was determined. Spaceflight and control cultures were chemically fixed 3 h and 3 d after launch. These fixed cultures were processed for scanning electron microscopy (SEM). The SEM analysis showed that with just 3 d of exposure to spaceflight, coverslip cultures contained 300 +/- 100 cells/mm2, whereas 1G control samples contained a confluent monolayer of cells (2400 +/- 200 cells/mm2). Although the cultures flown in space experienced a drastic decline in cell number in just 3 d, without further experimentation it was impossible to determine whether the decline was a result of microgravity, the harsh launch environment, or some combination of these factors. Therefore, this research attempted to address the effect of launch by subjecting osteoblasts to conditions simulating shuttle launch accelerations, noise, and vibrations. No differences, compared with controls, were seen in the number of total or viable cells after exposure to these various launch conditions. Taken together, these data indicate that the magnitude of gravitational loading (3G maximum) and vibration (7.83G rms maximum) resulting from launch does not adversely affect osteoblasts in terms of total or viable cell number immediately, but launch conditions, or the microgravity environment itself, may start a cascade of events that over several d contributes to cell loss.

NONSTEROIDAL ANTI-INFLAMMATORY DRUGS ARREST CELL CYCLE IN G0/G1PHASE AND INDUCE CELL DEATH IN OSTEOBLAST-ENRICHED CULTURES

Nonsteroidal anti-inflammatory drugs have been widely prescribed for orthopaedic patients to relieve pain and chronic inflammation. However, it has been demonstrated that NSAIDs suppress bone repair and remodeling in vivo. We have reported that ketorolac inhibits bone repair in vivo and its critical effective timing is at the early stage of endochondral ossification. Our previous results showed that ketorolac and indomethacin inhibit osteoblast proliferation in vitro, suggesting that this effect may be one of the mechanisms contributing to the suppressive effect of NSAIDs on bone remodeling. Cell proliferation and death of osteoblasts should be well regulated through some relative apoptotic and mitotic factors during normal bone remodeling process. Accordingly, we proposed that the induction of osteoblastic cell death of NSAIDs might be one of the mechanisms involving their suppressive effect on bone remodeling in vivo. In this study, we investigated whether NSAIDs arrest osteoblastic cell cycle and/or induce cell death. Whether the mechanism was mediated through prostaglandin (PG) pathway. We tested the effects of ketorolac, indomethacin, diclofenac, piroxicam on cell cycle kinetics, cytotoxicity, and cell death pattern in osteoblast-enriched cultures derived from fetal rat calvaria. Our results showed that ketorolac and indomethacin arrested cell cycle at G0/G1phase. All the 4 NSAIDs had cytotoxic effects and these effects were concentration dependent. The sequence of the cytotoxic effects of these four NSAIDs at 10-4M were indomethacin > diclofenac > ketorolac > piroxicam. Both PGE1and PGE2(10-10-10-8M) also significantly elevated the LDH leakage of osteoblasts, while PGF2αhad no significant effect. These results revealed that the cytotoxic effects of NSAIDs on osteoblasts might not be through inhibiting prostaglandin synthesis. They may be through PG-independent pathways. The results from flow cytometry followed by AnnexinV-FITC and propidium iodide double staining showed that 24 hours treatment of all the 4 NSAIDs (10-6and 10-4M) significantly induced both apoptosis (p<0.01) and necrosis (p<0.01, or p<0.05) in osteoblast cultures. These effects of NSAIDs on cell cycle arrest and cell death induction in osteoblasts may be one of the important mechanisms contributing to their suppressive effect on bone repair and bone remodeling in vivo.

Effects of nonsteroidal anti-inflammatory drugs and prostaglandins on osteoblastic functions

It has been reported that nonsteroidal anti-inflammatory drugs (NSAIDs) suppress bone repair and bone remodeling but only mildly inhibit bone mineralization at the earlier stage of the repair process. We proposed that the proliferation and/or the earlier stage of differentiation of osteoblasts may be affected by NSAIDs. This study was designed to investigate whether NSAIDs affect the proliferation and/or differentiation of osteoblasts and whether these effects are prostaglandin (PG) mediated. The effects of PGE 1 and PGE 2, indomethacin, and ketorolac on thymidine incorporation, cell count, intracellular alkaline phosphatase (ALP) activity, and Type I collagen content in osteoblast-enriched cultures derived from fetal calvaria were evaluated. The results showed that both PGs and NSAIDs inhibited DNA synthesis and cell mitosis in a time- and concentration-dependent manner. However, intracellular ALP activity and Type I collagen content were stimulated at an earlier stage of differentiation in osteoblasts. These results suggested that (i) the inhibitory effect of ketorolac on osteoblastic proliferation contributes to its suppressive effects on bone repair and remodeling in vivo; (ii) PGEs and NSAIDs may be involved in matrix maturation and biologic bone mineralization in the earlier stage of osteoblast differentiation; and (iii) the effects of ketorolac and indomethacin on cell proliferation and differentiation may not be through the inhibition of the synthesis of PGE 1 or PGE 2.

Effect of liposomes on mineralization in rat osteoblast-enriched cultures.

Liposomes, artificial membranous lipid vesicles, have been used as model structures for biological calcification processes. However, there is no definite conclusion that liposomes can be like matrix vesicles for inducing bone calcification and bone-like tissue formation on primary cultured cells. To determine whether liposomes can promote bone cell growth and mineralization by inducing crystal nucleation, liposomes composed of egg phosphatidylcholine, cholesterol, and bovine brain phosphatidylserine were added to 21-day-old Sprague-Dawley fetal rat calvarial cell cultures from day 1. The aims were to observe proliferation and the phenotype of osteoblasts by measuring cell numbers and alkaline phosphatase (ALP) activity and, when added at confluence, to observe calcification. The data were analyzed with two-way ANOVA. During the 16-day culture period, cell numbers were not significantly affected by liposomes (100 mumol/L). However, ALP activity was significantly inhibited by liposomes (p < 0.05) at day 16 and thereafter. Calcified particles were detected by von Kossa's method, and were larger and more abundant (p < 0.05) in the liposomes groups than in the control from days 12-24. This response depended on liposomes dose. These findings suggest that liposomes promote calcification and accelerate the formation of bone-like tissue. Liposomes slightly reduce the expression of the osteoblast phenotype and do not affect cell growth in primary rat osteoblast-enriched cultures.

Alternate signaling pathways selectively regulate binding of insulin-like growth factor I and II on fetal rat bone cells

Bone cells synthesize and respond to IGF-I and IGF-II which contribute to bone remodeling and linear growth. In osteoblasts, prostaglandin (PG)E2 stimulates IGF-I but not IGF-II synthesis through a cAMP-dependent protein kinase A (PKA)-related event. However, protein kinase C (PKC) activation by PGE2 enhances replication and protein synthesis by less differentiated periosteal cells more so than in osteoblast-enriched cultures from fetal rat bone. Using various PGs and other PKA and PKC pathway activators, the importance of these aspects of PGE2 activity has now been examined on IGF receptors in these bone cell culture models. PGE2 and other agents that activate PKA enhanced 125I-IGF-II binding to type 2 IGF receptors on both cell populations. In contrast, agents that activate PKC enhanced 125I-IGF-I binding to type 1 receptors on less differentiated bone cells, and of these, only phorbol myristate acetate (PMA), which activates PKC in a receptor-independent way, was effective in osteoblast-enriched cultures. No stimulator increased total type 1 receptor protein in either cell population. Consequently, ligand binding to type 1 and type 2 IGF receptors is differentially modulated by specific intracellular pathways in bone cells. Importantly, changes in apparent type 1 receptor number occur rapidly and may do so at least in part through post-translational effects. These results may help to predict new ways to manipulate autocrine or paracrine actions by IGFs in skeletal tissue.

Opposing effects by glucocorticoid and bone morphogenetic protein-2 in fetal rat bone cell cultures

Glucocorticoid in excess produces bone loss in vivo. Consistent with this, it reduces the stimulatory effect of transforming growth factor beta (TGF-beta) on collagen synthesis in osteoblast-enriched cultures in vitro, where it also suppresses TGF-beta binding to its type I receptors. Analogous studies with bone morphogenetic protein-2 (BMP-2) show directly opposite results. These findings prompted us to assess the effect of glucocorticoid on BMP-2 activity in cultured bone cells, and whether either agent had a dominant influence on TGF-beta binding or function. BMP-2 activity was retained in part in osteoblast-enriched cultures pre-treated or co-treated with cortisol, and was fully evident when glucocorticoid exposure followed BMP-2 treatment. In addition, BMP-2 suppressed the effects of cortisol on TGF-beta activity, on TGF-beta binding, and on gene promoter activity directed by a glucocorticoid sensitive transfection construct. While BMP-2 also alters the function of less-differentiated bone cells, it only minimally prevented cortisol activity in these cultures. Our studies indicate that BMP-2 can oppose certain effects by cortisol on differentiated osteoblasts, and may reveal useful ways to diminish glucocorticoid-dependent bone wasting.

Rapid Flux in Transforming Growth Factor-β Receptors on Bone Cells

The proportion of transforming growth factor-beta (TGF-beta) binding among conventional membrane receptors on bone cells can vary with hormone or growth factor treatment or with the state of osteoblast-like activity and appears to determine the nature of its biological effects. Therefore, functional TGF-beta receptor stability could be an important aspect of regulation. Suppression of protein synthesis reduced TGF-beta binding to types I and II receptors with t1/2 of 2 h and to betaglycan with t1/2 of 6 h. In contrast, suppression of mRNA transcription reduced TGF-beta binding at least 3-fold more slowly at each receptor site. Preexposure to TGF-beta decreased its binding at all three sites within 4 h in osteoblast-enriched cultures. This effect was transient with lower TGF-beta concentrations, where the receptor profile was nearly fully restored within 24-48 h. In contrast, less differentiated bone cells were less sensitive to ligand-dependent receptor down-regulation. Agents that alter protein kinase and phosphatase activity also modified the TGF-beta binding profile in specific ways. Together, these results indicate that cell surface TGF-beta receptors turn over rapidly by ligand-independent and ligand-dependent mechanisms, demonstrate that the binding capacity of TGF-beta receptors is less stable than their mRNAs, and that functional receptor levels may be determined in part by post-transcriptional events.

Expression of insulin-like growth factor I (IGF-I) mRNA variants in rat bone.

The rat insulin-like growth factor I (IGF-I) gene is characterized by the presence of multiple mRNA transcripts, which differ in the 5'- and/or 3'-untranslated regions (UTRs). Transcript initiation occurs in either exon 1 or exon 2, giving rise to mRNA species which differ in the length and sequence of the 5'-UTR, while further variation is due to multiple transcription start sites and differential splicing within exon 1. This heterogeneity is indicative of multifaceted regulation of gene expression, and it is likely that differences in the nature of transcript expression reflect cell-specific regulation. As IGF-I is an important factor in skeletal growth and development, the aim of this study was to determine the pattern of transcript expression in rat whole bone and osteoblast-enriched cultures isolated from long bones. The relative proportions of transcripts differing in the 5'-UTR were determined by RNase protection assays and compared to expression in rat liver. These studies revealed a significantly lower expression of exon 2-derived transcripts in bone cells compared to liver (approximately 10% compared to 40% of total transcripts). There were also important differences in start site usage in exon 1 in bone cells. In osteoblastic cells, transcripts initiated at start site 3 were the predominant species (50% +/- 12% of total exon 1-derived mRNAs; Mean +/- SD) whereas the alternately spliced transcripts represented only 20% +/- 3%. This was in contrast to the profile in liver in which 47% +/- 9% of total exon 1-derived transcripts were the alternately spliced mRNAs, but start site 3-initiated transcripts represented only 11% +/- 3%. In addition, the proportion of transcripts initiated at start site 4 was about twofold greater in liver than in bone cells (32% +/- 7% compared to 16% +/- 8%). However, expression of full-length transcripts was similar in both tissues. The distribution in osteoblastic cells reflected that in whole bone. These results demonstrate that the IGF-I transcript profile in bone cells differs to that in liver cells. Since the mRNA variants exhibit different properties, including half-life and translatability, such cell-specific variation in their relative expression is likely to reflect differential regulation of IGF-I in these tissues.

Microvessel endothelial cells and pericytes increase proliferation and repress osteoblast phenotypic markers in rat calvarial bone cell cultures

To investigate the influence of microvessel cells on osteoblasts, we exposed osteoblast-enriched cultures of rat calvarial cells to cultured endothelial cells and pericytes using feeder-layer co-cultures, co-culture dish inserts, and conditioned media experiments. When co-cultured with growth-arrested feeder-layers of endothelial cells or pericytes for 10 days, bone cell cultures showed an increase in cell number and reduction in alkaline phosphatase activity. The response of bone cells to endothelial cells was nearly twice their response to pericytes. A similar response was demonstrated by exposure to microvessel cells in co-culture dish inserts and by exposure to media conditioned by microvessel cells. In long-term cultures of bone cells, the levels of osteocalcin and the number of mineralized nodules both were reduced by exposure to media conditioned by the microvessel cells. Transient exposure to conditioned media from the microvessel cell cultures for 3 days, during the period from initial plating to cell confluence, produced nearly the same effect on the cultures of bone cells as did continuous exposure to these conditioned media. The influence of isolated microvessel cells on osteoblast-enriched calvarial cells was found to be primarily mitogenic, mediated by soluble factors, independent of cell contact, and a cause of prolonged reduction in the expression of early and late markers of the osteoblast phenotype.

Independent changes in type I and type II receptors for transforming growth factor beta induced by bone morphogenetic protein 2 parallel expression of the osteoblast phenotype.

Transforming growth factor beta (TGF-beta), a potent regulator of bone formation, has bifunctional effects on osteoblast replication and biochemical activity that appear differentiation dependent. We now show that cell surface binding sites for TGF-beta vary markedly among fibroblasts, bone-derived cells, and highly differentiated osteosarcoma cultures from fetal rats. Expression of betaglycan and type II receptors decline relative to type I receptor expression in parallel with an increase in osteoblast-like activity, predicting that the ratio among various TGF-beta binding sites could influence how its signals are perceived. Bone morphogenetic protein 2 (BMP-2), which induces osteoblast function, does not alter TGF-beta binding or biochemical activity in fibroblasts and has only small effects in less differentiated bone cells. In contrast, BMP-2 rapidly reduces TGF-beta binding to betaglycan and type II receptors in osteoblast-enriched primary cell cultures and increases its relative binding to type I receptors in these cells and in ROS 17/2.8 cultures. Pretreatment with BMP-2 diminishes TGF-beta-induced DNA synthesis in osteoblast-enriched cultures but synergistically enhances its stimulatory effects on either collagen synthesis or alkaline phosphatase activity, depending on the present state of bone cell differentiation. Therefore, BMP-2 shifts the TGF-beta binding profile on bone cells in ways that are consistent with progressive expression of osteoblast phenotype, and these changes distinguish the biochemical effects mediated by each receptor. Our observations indicate specific stepwise actions by TGF-beta family members during osteoblast differentiation, developing in part from changes imprinted by BMP-2 on TGF-beta receptor stoichiometry.

Cortisol downregulates osteoblast α1(I) procollagen mRNA by transcriptional and posttranscriptional mechanisms

Glucocorticoids decrease osteoblast proliferation and type I collagen production, and this may play a role in the development of glucocorticoid-induced osteoporosis. Osteoblast-enriched cultures derived from fetal rat calvaria were used to determine the mechanisms by which cortisol decreases alpha 1 (I) procollagen expression in bone cells. A 24 h treatment with cortisol decreased collagen synthesis in these cultures in a dose-dependent manner. Cortisol decreased alpha 1 (I) procollagen transcripts in a dose- and time-dependent manner as well. Repression of alpha 1 (I) procollagen transcripts was evident as early as 2 h of treatment and was maximal after 48 h of treatment. Nuclear run-off assays showed that cortisol downregulated transcription of the alpha 1 (I) procollagen gene. In addition, pretreatment with cortisol decreased the stability of alpha 1 (I) procollagen mRNA in transcription-arrested osteoblast cultures. The ability of cortisol to downregulate alpha 1 (I) procollagen transcripts was sensitive to cycloheximide treatment, suggesting that the gene is under "secondary control" by glucocorticoids. Since cortisol decreases alpha 1 (I) procollagen gene transcription in osteoblasts but does not affect alpha 1 (I) procollagen gene transcription in fibroblasts, we suggest that the mechanisms controlling glucocorticoid repression of collagen expression are cell-type specific.

TGF-β and PDGF Act Synergistically in Affecting the Growth of Human Osteoblast-Enriched Cultures

Transforming growth factor beta (TGF-beta) is a polypeptide found in high concentrations in bone and is produced by and acts on primary adult human derived osteoblast-enriched cultures (PHO cells). Receptors for TGF-beta are present on PHO cells and TGF-beta is mitogenic for these cells. Results of these studies in conjunction with those of others suggest that TGF-beta may have an important therapeutic role in orthopaedic surgery; however, with respect to its mitogenic actions, further studies were needed to establish whether TGF-beta was acting directly to stimulate the growth of PHO cells. TGF-beta has been found in other systems to act as an indirect mitogen, stimulating growth via secretion of another growth factor, platelet-derived growth factor (PDGF). In an effort to determine whether the TGF-beta growth stimulation was mediated directly or indirectly, we have examined the growth stimulation of PHO cells by PDGF alone and in combination with TGF-beta. These studies revealed that TGF-beta in combination with either PDGF-AA or BB led to stimulation greater than that observed with either growth factor alone. TGF-beta in combination with PDGF-BB led to a synergistic stimulatory response while that observed with the AA isoform was more nearly additive. Further studies demonstrated that TGF-beta was capable of up-regulating the protein levels of the PDGF alpha (alpha) receptor within thirty minutes of TGF-beta pretreatment. Thus, TGF-beta appears to have both direct and indirect mechanisms of action as a mitogen in the PHO system. Finally, we showed that both the positive and negative alkaline phosphatase staining PHO cells were responsive to the mitogenic effects of both growth factor singly and in combination.

Complex pattern of insulin-like growth factor binding protein expression in primary rat osteoblast enriched cultures: regulation by prostaglandin E2, growth hormone, and the insulin-like growth factors.

Primary osteoblast-enriched (Ob) cultures from fetal rat bone synthesize insulin-like growth factor (IGF) I and IGF-II, which each enhance Ob function. While a number of agents modulate IGF-I production, IGF-II is constitutively expressed in this culture model. Independent of their expression, however, the activity of the IGFs can be modified by a small group of proteins termed IGF binding proteins (IGFBPs), but little is known about the regulation of individual IGFBPs that are synthesized by Ob cells. Northern blot analysis revealed that serum-deprived primary rat Ob cells express transcripts encoding IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6, but undetectable levels of IGFBP-1 transcripts. Western ligand blots of Ob culture medium probed with 125I-IGF-I or 125I-IGF-II showed predominant IGFBPs migrating at 30/32 kDa, with minor bands at 24 and 38-47 kDa. Western antibody analysis identified IGFBP-2 and IGFBP-5 within the 30/32 kDa complex, while gel mobility shift on SDS-PAGE following deglycosylation determined that IGFBP-3 comprised the 38-47 kDa complex. By Northern analysis, 6 h treatment with prostaglandin E2 (PGE2), growth hormone (hGH), IGF-I, or IGF-II revealed a complex pattern of regulatory effects on steady-state IGFBP transcript expression. PGE2 increased the transcript levels of IGFBP-3, IGFBP-4, and IGFBP-5, (approximately 22-, approximately 2- and approximately 4-fold respectively), but had no effect on IGFBP-2 or IGFBP-6 transcripts. hGH enhanced IGFBP-3 and IGFBP-5 transcripts (each approximately twofold). IGF-I and IGF-II had no effect on IGFBP-2 steady-state transcript levels but enhanced the level of IGFBP-5 transcripts (approximately fourfold). By Western ligand blot analysis, 24 h treatment with PGE2 elevated the 24 and 38-47 kDa IGFBPs and to a lesser extent the 30/32 kDa complex, hGH elevated the 38-47 kDa IGFBPs, and IGF-I and IGF-II each increased the 30/32 kDa IGFBP complex. Therefore, a comparison of results obtained from Northern, Western ligand, and Western antibody studies indicates that multiple IGFBPs are expressed by primary rat Ob cultures. While IGFBP-2 and IGFBP-6 synthesis in Ob cultures is relatively unaffected by short-term treatment with PGE2, hGH, or the IGFs, these agents modify IGFBP-3, IGFBP-4, and IGFBP-5 expression with individual patterns of effects. In addition, some changes in IGFBP polypeptide levels that are independent of alterations in transcript expression may result from the formation of complexes between IGFs and certain IGFBPs, which could serve to store IGFs for future utilization in the formation phase of bone remodeling.

Oxygen tension regulates osteoblast function

Abrupt changes in oxygen availability within the periodontium have been suggested to have a regulatory role in alveolar bone remodeling during tooth movement; arguably, similar to that seen in bone growth or fracture healing. The purpose of this investigation was therefore to study the effects of ambient hypoxia and hyperoxia on osteoblast function in vitro. Osteoblast-enriched cultures from fetal rat calvariae were exposed to atmospheres of hyperoxia (90% O 2) and hypoxia (10% O 2) and assayed for media pH, PO 2, pCO 2, cellular proliferation, alkaline phosphatase (AP) activity, and collagen synthesis. Results of this study show that in low ambient oxygen tension cellular proliferation increases, whereas the AP activity, collagen synthesis, media PO 2, PCO 2 decreases. In contrast, in hyperoxic conditions cellular proliferation is suppressed with concomitant increases in: AP activity, collagen synthesis, and partial pressures for oxygen and carbon dioxide. Media pH remained unaffected. In crossover experiments, where cells were initially grown in hypoxic conditions and were switched to hyperoxic conditions, their metabolic activities were abruptly reversed. These findings in conjunction with earlier reports, suggest a triggering role for oxygen tension (an environmental factor) in bone remodeling. (A M J O RTHOD D ENTOFAC O RTHOP 1994;105:457-63.)

Prostaglandin E2 rapidly stimulates insulin-like growth factor-I gene expression in primary rat osteoblast cultures: evidence for transcriptional control.

Osteoblast-enriched (Ob) cultures isolated from fetal rat bone synthesize insulin-like growth factor-I (IGF-I), which functions as a locally acting growth and differentiation factor in the skeleton. Consistent with prior studies demonstrating that IGF-I production is enhanced in bone by agents that induce cAMP, prostaglandin E2 (PGE2) stimulates both cAMP synthesis and IGF-I mRNA in Ob cells. However, little is known about how cAMP regulates IGF-I expression in this or any other cell system. In rat tissues, multiple mechanisms influence levels of IGF-I mRNA, including transcription from two promoters, differential RNA splicing and stability, and alternative RNA polyadenylation. To determine how cAMP influences IGF-I gene expression in Ob cultures, we examined the responses of these cells to treatment with PGE2. PGE2 rapidly enhanced the accumulation of both large and small IGF-I transcripts, with increases in IGF-I mRNA detected within 2 h of treatment and persisting for 24 h. Analysis of precursor RNA by a highly specific and sensitive ribonuclease protection assay demonstrated a rise in nascent IGF-I mRNA within 30 min of exposure to PGE2, with a peak stimulation of 4-fold above control levels seen by 2 h and levels remaining elevated for up to 24 h. IGF-I transcripts in Ob cells were directed only by promoter 1, the more 5' of the two rat IGF-I gene promoters. As additionally assessed using the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole, PGE2 treatment had little effect on IGF-I mRNA stability. In aggregate, these studies show that in fetal rat Ob cultures, PGE2 enhances IGF-I gene expression primarily through transcriptional mechanisms that are limited to a single IGF-I gene promoter. Ob cells, therefore, may be an excellent model for determining how cAMP regulates IGF-I gene transcription.