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M Centrella

Publications and source records attributed to M Centrella.

At least 37 records · Page 2Linked to original sources
PubMed1997

Parathyroid hormone-related protein enhances insulin-like growth factor-I expression by fetal rat dermal fibroblasts.

Interactions between cells of differing embryonic origins comprise a common theme during tissue development and repair. Often, communication between them can be mediated by soluble growth mediators and in some cases is restricted in focus. That is, some cells respond to, but do not produce, mediators expressed by other cells within the tissue. Because keratinocytes respond to but do not express insulin-like growth factor I (IGF-I), another skin cell population, the dermal fibroblast, may supply this factor. However, keratinocytes express, but do not respond to parathyroid hormone related protein (PTHrp), which increases cAMP production by dermal fibroblasts. Based on earlier results where inducers of cAMP increase local IGF-I expression in skeletal tissue, we postulated that PTHrp might induce local IGF-I by dermal fibroblasts and provide a source of this factor for keratinocyte activity. Our studies reveal that IGF-I mRNA and protein levels increase in response to PTHrp in vitro, and that this effect is replicated by inducers of cAMP, but not by activators of protein kinase C. Consequently, these factors appear to comprise a paracrine loop within the skin, permitting focused but restricted IGF-I expression to support skin growth, remodeling, or repair.

Animals↗
PubMed1997

Multiple and essential Sp1 binding sites in the promoter for transforming growth factor-beta type I receptor.

Maximal gene expression driven by the promoter for the transforming growth factor beta type I receptor (TGF-betaRI) occurs with a 1. 0-kilobase pair fragment immediately upstream of exon 1. This region lacks a typical TATA box but contains CCAAT boxes, multiple Sp1, and PEBP2/CBFalpha binding sites among other possible cis-acting elements. Alterations within two CCAAT box sequences do not mitigate reporter gene expression driven by the basal promoter, and no nuclear factor binds to oligonucleotides encompassing these sites. In contrast, other deletions or site-specific mutations reveal an essential Sp1 site in the basal promoter and several dispersed upstream Sp1 sites that contribute to maximal reporter gene expression. The proportions of transcription factors Sp1 and Sp3, and their ratios of binding to consensus elements, are maintained in bone cells at different stages of differentiation. Finally, nuclear factor that binds to PEBP2/CBFalpha-related cis-acting elements in the basal promoter sequence also occurs in osteoblasts. Our studies reveal that constitutive expression of TGF-betaRI may be determined by constitutive nuclear factor binding to Sp1 sites, whereas other elements may account for the variations in TGF-betaRI levels that parallel changes in bone cell differentiation or activity.

Activin Receptors, Type I↗
PubMed1997

17beta-estradiol potently suppresses cAMP-induced insulin-like growth factor-I gene activation in primary rat osteoblast cultures.

Insulin-like growth factor-I (IGF-I) is a key factor in bone remodeling. In osteoblasts, IGF-I synthesis is enhanced by parathyroid hormone and prostaglandin E2 (PGE2) through cAMP-activated protein kinase. In rats, estrogen loss after ovariectomy leads to a rise in serum IGF-I and an increase in bone remodeling, both of which are reversed by estrogen treatment. To examine estrogen-dependent regulation of IGF-I expression at the molecular level, primary fetal rat osteoblasts were co-transfected with the estrogen receptor (hER, to ensure active ER expression), and luciferase reporter plasmids controlled by promoter 1 of the rat IGF-I gene (IGF-I P1), used exclusively in these cells. As reported, 1 microM PGE2 increased IGF-I P1 activity by 5-fold. 17beta-Estradiol alone had no effect, but dose-dependently suppressed the stimulatory effect of PGE2 by up to 90% (ED50 approximately 0.1 nM). This occurred within 3 h, persisted for at least 16 h, required ER, and appeared specific, since 17alpha-estradiol was 100-300-fold less effective. By contrast, 17beta-estradiol stimulated estrogen response element (ERE)-dependent reporter expression by up to 10-fold. 17beta-Estradiol also suppressed an IGF-I P1 construct retaining only minimal promoter sequence required for cAMP-dependent gene activation, but did not affect the 60-fold increase in cAMP induced by PGE2. There is no consensus ERE in rat IGF-I P1, suggesting novel downstream interactions in the cAMP pathway that normally enhances IGF-I expression in skeletal cells. To explore this, nuclear extract from osteoblasts expressing hER were examined by electrophoretic mobility shift assay using the atypical cAMP response element in IGF-I P1. Estrogen alone did not cause DNA-protein binding, while PGE2 induced a characteristic gel shift complex. Co-treatment with both hormones caused a gel shift greatly diminished in intensity, consistent with their combined effects on IGF-I promoter activity. Nonetheless, hER did not bind IGF-I cAMP response element or any adjacent sequences. These results provide new molecular evidence that estrogen may temper the biological effects of hormones acting through cAMP to regulate skeletal IGF-I expression and activity.

Animals↗
PubMed1997

Effect of strain on human keratinocytes in vitro.

Tissue expansion, a technique to enlarge the skin surface area with an expandable balloon, has been widely used in reconstructive surgery. Although the effect of tissue expansion on in vivo skin physiology and histology has been well documented, it remains unclear whether keratinocytes or other cell types are responsible for these changes. Therefore, we investigated the in vitro effect of cyclic (10 cycles/min, 150 mmHg) or constant (continuous, 150 mmHg) strain on human keratinocyte phenotype and relevant mechanosignaling pathways. Our results demonstrate that keratinocytes subjected to cyclic strain exhibit a significant (P < 0.05) increase in cell proliferation (49.2+/-15.8%), DNA synthesis (37.7+/-4.5%), elongation (20.3+/-2.7%), and protein synthesis (17.9+/-6.6% increase) as compared with stationary controls. In contrast, keratinocytes subjected to constant strain were unaffected aside from a modest transitory increase in the proliferative rate. Keratinocytes subjected to cyclic strain aligned perpendicular to the force vector (24.2+/-1.6 degrees) as compared with stationary controls (40.4+/-2.2 degrees; the smaller degree indicates better alignment). We also report strain-induced reduction in the levels of cyclic adenosine mono phosphate (cAMP), protein kinase A (PKA), and prostaglandin E2 (PGE2) as compared with stationary controls (cAMP, 30+/-7.5%; PKA, 45+/-17%; PGE2, 58+/-4.3%; percent decrease vs. that of control). We conclude that direct application of cyclic strain on human keratinocytes modulates cell phenotype and cAMP-mediated signaling pathways in an inverse manner. Moreover, keratinocytes may play an important role in previously observed alterations in skin properties associated with tissue expansion and other strain-induced responses.

Cell Division↗
PubMed1996

Cloning, characterization, and expression of the transforming growth factor-beta type I receptor promoter in fetal rat bone cells.

Transforming growth factor (TGF-beta) binds several discrete membrane proteins. Of these, a type 1 receptor appears indispensable for signal transduction. Previous examination of TGF-beta receptor expression has been limited to changes in cell surface protein, and more recently, mRNA abundance. In order to learn more about TGF-beta function and receptor expression during osteogenesis, we have now cloned a 4 kilobase (kb) DNA fragment 5' proximal to the coding region of the rat TGF-beta type I receptor gene. Sequence analysis revealed multiple elements compatible with transcription initiation, including a properly positioned and oriented CCAAT box, six Sp1 binding sites (three defining GC boxes), and two strong AP2 binding sites within a 0.7 kb span directly upstream of the coding region. The 3' terminal 0.3 kb span comprises a GC-enriched (77%) so-called CpG island that, like other similarly organized promoters, lacks a TATA box. Primer extension and RNase protection studies with cRNAs from this area show multiple initiation sites within 220 bp 5' proximal to the initial methionine codon. Transient transfections using nested, deleted, and inverted promoter sequences demonstrated maximal reporter expression by a 1 kb fragment encompassing all of these elements. Truncation of the 1 kb fragment from the 5' and 3' ends indicated the need for several elements for peak promoter activity. These results, and transfections in fetal rat bone and dermal cells, suggest that this promoter contains elements that specify basal and conditional expression of the TGF-beta type I receptor in bone.

Amino Acid Sequence↗
PubMed1996

Identification of the cAMP response element that controls transcriptional activation of the insulin-like growth factor-I gene by prostaglandin E2 in osteoblasts.

Insulin-like growth factor-I (IGF-I), a multifunctional growth factor, plays a key role in skeletal growth and can enhance bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other agents that increase cAMP activated IGF-I gene transcription in primary rat osteoblast cultures through promoter 1 (P1), the major IGF-I promoter, and found that transcriptional induction was mediated by protein kinase A. We now have identified a short segment of P1 that is essential for full hormonal regulation and have characterized inducible DNA-protein interactions involving this site. Transient transfections of IGF-I P1 reporter genes into primary rat osteoblasts showed that the 328-base pair untranslated region of exon 1 was required for a full 5.3-fold response to PGE2; mutation in a previously footprinted site, HS3D (base pairs +193 to +215), reduced induction by 65%. PGE2 stimulated nuclear protein binding to HS3D. Binding, as determined by gel mobility shift assay, was not seen in nuclear extracts from untreated osteoblast cultures, was detected within 2 h of PGE2 treatment, and was maximal by 4 h. This DNA-protein interaction was not observed in cytoplasmic extracts from PGE2-treated cultures, indicating nuclear localization of the protein kinase A-activated factor(s). Activation of this factor was not blocked by cycloheximide (Chx), and Chx did not impair stimulation of IGF-I gene expression by PGE2. In contrast, binding to a consensus cAMP response element (CRE; 5'-TGACGTCA-3') from the rat somatostatin gene was not modulated by PGE2 or Chx. Competition gel mobility shift analysis using mutated DNA probes identified 5'-CGCAATCG-3' as the minimal sequence needed for inducible binding. All modified IGF-I P1 promoterreporter genes with mutations within this CRE sequence also showed a diminished functional response to PGE2. These results identify the CRE within the 5'-untranslated region of IGF-I exon 1 that is required for hormonal activation of IGF-I gene transcription by cAMP in osteoblasts.

Animals↗
PubMed1996

Rapid flux in transforming growth factor-beta 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.

Animals↗
PubMed1996

Promoter-dependent and -independent activation of insulin-like growth factor binding protein-5 gene expression by prostaglandin E2 in primary rat osteoblasts.

Insulin-like growth factor (IGF) action is mediated by high affinity cell surface IGF receptors and modulated by a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of six IGFBPs characterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E2 (PGE2), a local factor that mediates certain effects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain. In this study, we show that transcriptional and post-transcriptional events initiated by PGE2 collaborate to enhance IGFBP-5 gene expression in primary fetal rat osteoblast cultures. PGE2 treatment stimulated up to a 7-fold rise in steady-state levels of IGFBP-5 mRNA throughout 32 h of incubation. Analysis of nascent IGFBP-5 mRNA suggested that PGE2 had only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter-reporter genes confirmed that PGE2 enhanced promoter activity by approximately 2-fold. Similar stimulatory effects were seen with forskolin. A DNA fragment with only 51 base pairs of the 5'-flanking sequence retained hormonal responsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts with the mRNA transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that PGE2 enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t1/2 from 9 to 18 h. The effects of PGE2 on steady-state IGFBP-5 transcripts were abrogated by preincubating cells with cycloheximide, indicating that the effects of PGE2 on both gene transcription and mRNA stability required ongoing protein synthesis. Therefore, both promoter-dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE2 in osteoblasts.

Animals↗
PubMed1996

Modulation of transforming growth factor beta receptor levels on microvascular endothelial cells during in vitro angiogenesis.

Microvascular endothelial cells (RFCs) cultured in two-dimensional (2D) cultures proliferate rapidly and exhibit an undifferentiated phenotype. Addition of transforming growth factor beta1 (TGFbeta1) increases fibronectin expression and inhibits proliferation. RFCs cultured in three-dimensional (3D) type I collagen gels proliferate slowly and are refractory to the anti-proliferative effects of TGF beta1. TGF beta1 promotes tube formation in 3D cultures. TGF beta1 increases fibronectin expression and urokinase plasminogen activator (uPA) activity and plasminogen activator inhibitor-1 (PAI-1) levels in 3D cultures. Since the TGF beta type I and II receptors have been reported to regulate different activities induced by TGF beta1, we compared the TGF beta receptor profiles on cells in 2D and 3D cultures. RFCs in 3D cultures exhibited a significant loss of cell surface type II receptor compared with cells in 2D cultures. The inhibitory effect of TGF beta1 on proliferation is suppressed in transfected 2D cultures expressing a truncated form of the type II receptor, while its stimulatory effect on fibronectin production is reduced in both 2D and 3D transfected cultures expressing a truncated form of the type I receptor. These data suggest that the type II receptor mediates the antiproliferative effect of TGF beta1 while the type I receptor mediates the matrix response of RFCs to TGF beta1 and demonstrate that changes in the matrix environment can modulate the surface expression of TGF beta receptors, altering the responsiveness of RFCs to TGF beta1.

Animals↗
PubMed1996

Regulation of proliferative response of cardiac fibroblasts by transforming growth factor-beta 1.

Cardiac fibroblasts constitute greater than 90% of the non-myocyte cells in the heart. Previously, it was established that cardiac fibroblasts are predisposed to transformation into a phenotype with muscle-specific features and that transforming growth factor-beta 1 (TGF-beta 1) is a specific inducer of this event. In this study the hypothesis that TGF-beta 1-induced phenotypic modulation of cardiac fibroblasts is associated with their altered proliferative capacity is tested. Therefore the effects of TGF-beta 1 on DNA synthesis in cardiac fibroblasts under normal conditions of cell culture and in response to a potent mitogen, basic fibroblasts growth factor (bFGF) were determined. The results showed that TGF-beta 1 at 15 ng/ml (a concentration that induces fibroblast "transformation") had a regulatory effect on proliferative capacity of cardiac fibroblasts which varied as the function of cell density in culture. In subconfluent and confluent cultures, pre-treatment of cardiac fibroblasts with TGF-beta 1 for 24 h resulted in a dramatic shift in the bFGF-induced stimulation of DNA synthesis. TGF-beta 1-induced inhibition of DNA synthesis in cardiac fibroblasts coincided with their phenotypic modulation as evidenced by the expression of sarcomeric actin mRNA and morphological changes. Cross-linking studies with [125I]-labeled TGF-beta 1 showed the presence of conventional types I, II and III TGF-beta 1 receptor complexes on cardiac fibroblasts and their binding to TGF-beta 1 under the experimental conditions. In summary, these data indicate that the proliferative capacity of cardiac fibroblasts is controlled by TGF-beta 1. They further suggest that the TGF-beta 1-induced phenotypic modulation of cardiac fibroblasts may be extended to include their altered proliferative capacity.

Actins↗
PubMed1996

Oncostatin-M: a new bone active cytokine that activates osteoblasts and inhibits bone resorption.

Osteoblasts and their precursors respond to specific cytokines, growth factors, and hormones. One facet of this response includes the secretion of additional cytokines, some of which are part of the circuitry involved in the regulation of osteoblast and osteoclast function. Therefore, understanding which cytokines are able to activate osteoblastic cells and the consequences of that activation are central to understanding normal and pathologic bone remodeling. Oncostatin M (OSM) is a glycoprotein belonging to a new subfamily of cytokines related by sequence and structural homology and the use of the signal transducing receptor component gp130. Osteoblastic cells secrete and respond to leukemia-inhibiting factor (LIF) both in vitro and in vivo, suggesting that LIF is an autocrine regulatory factor. OSM is closely related to LIF, and therefore we hypothesized that OSM should regulate the function of cells in the osteoblastic lineage. Primary neonatal murine or fetal rat calvarial osteoblastic cultures were treated with OSM or LIF and a series of biochemical and biological parameters were determined. In these cultures, OSM induced proliferation, collagen synthesis, and interleukin-6 secretion, whereas it inhibited alkaline phosphatase activity. Bone resorption was also inhibited by OSM. These data represent the first report of OSM's effects on bone cell function and indicate that, like some other members of the LIF/interleukin-6 subfamily, OSM has potent bone regulatory activity.

Alkaline Phosphatase↗
PubMed1995

Expression of transforming growth factor type III receptor in vascular endothelial cells increases their responsiveness to transforming growth factor beta 2.

Bovine aortic endothelial cells (BAECs) express both type I and type II receptors for transforming growth factor beta (TGF beta). These cells respond to TGF beta 1 but are relatively refractory to another isoform of TGF beta, termed TGF beta 2. TGF beta s are thought to signal through receptor complexes composed of type I and/or type II receptors, both of which appear to be functional serine-threonine kinases. The TGF beta type III receptor, on the other hand, does not seem to have any direct signaling capacity. We have now stably transfected BAECs with the type III receptor cDNA. These cells displayed surface expression of the type III receptor protein, as determined by cross-linking with iodinated TGF beta 1 and immunoprecipitation with antibodies to the type III receptor protein. Transfected BAECs exhibit increased responsiveness to TGF beta 2 by several different criteria including an increase in plasminogen activator inhibitor-1 protein and inhibition of migration and proliferation. Thus, the type III receptor protein may play a role in presenting TGF beta 2 to the type II receptor and increase responsiveness to TGF beta 2 to a level comparable to that of TGF beta 1.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

Regulation of insulin-like growth factor I transcription by cyclic adenosine 3',5'-monophosphate (cAMP) in fetal rat bone cells through an element within exon 1: protein kinase A-dependent control without a consensus AMP response element.

Insulin-like growth factor I (IGF-I) is a locally synthesized anabolic growth factor for bone. IGF-I synthesis by primary fetal rat osteoblasts (Ob) is stimulated by agents that increase the intracellular cAMP concentration, including prostaglandin E2 (PGE2). Previous studies with Ob cultures demonstrated that PGE2 enhanced IGF-I transcription through selective use of IGF-I promoter 1, with little effect on IGF-I messenger RNA half-life. Transient transfection of Ob cultures with an array of promoter 1-luciferase reporter fusion constructs has now allowed localization of a potential cis-acting promoter element(s) responsible for cAMP-stimulated gene expression to the 5'-untranslated region (5'-UTR) of IGF-I exon 1, within a segment lacking a consensus cAMP response element. Our evidence derives from three principal observations: 1) a transfection construct containing only 122 nucleotides (nt) of promoter 1 and 328 nt of the 5'-UTR retained full PGE2-stimulated reporter expression; 2) maximal PGE2-driven reporter expression required the presence of nt 196 to 328 of exon 1 when tested within the context of IGF-I promoter 1; 3) cotransfection of IGF-I promoter-luciferase-reporter constructs with a plasmid encoding the alpha-isoform of the catalytic subunit of murine cAMP-dependent protein kinase (PKA) produced results comparable to those seen with PGE2 treatment, whereas cotransfection with a plasmid encoding a mutant regulatory subunit of PKA that cannot bind cAMP blocked PGE2-induced reporter expression. Deoxyribonuclease I footprinting of the 5'-UTR of exon 1 demonstrated protected sequences at HS3A, HS3B, and HS3D, three of six DNA-protein binding sites previously characterized with rat liver nuclear extracts. Of these three regions, only the HS3D binding site is located within the functionally identified hormonally responsive segment of IGF-I exon 1. These results directly implicate PKA in the control of IGF-I gene transcription by PGE2 and identify a segment of IGF-I exon 1 as being essential for this hormonal regulation.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

Differential actions of prostaglandins in separate cell populations from fetal rat bone.

Prostaglandins (PGs) may stimulate or inhibit bone cell replication and protein synthesis. These disparities may be concentration or time dependent, or occur in discrete cell types or by different second signals. Cell populations that express progressive degrees of osteoblast-like activity can be obtained by serial collagenase digestion of fetal rat parietal bone. The first (population 1) appears less differentiated, whereas the later (populations 3-5) exhibit biochemical features characteristic of osteoblasts. Within 24 h of treatment, three separate PGs increased DNA synthesis in population 1 with relative potencies of PGE1 < PGE2 < PGF2 alpha. By contrast, PGE1 and PGE2 (both strong cAMP inducers) inhibited basal DNA synthesis in population 3-5. These differences were paralleled by analogous changes in collagen and noncollagen synthesis in each population. The mitogenic effect in population 1 persisted for 72 h, and at later times was sensitive to indomethacin. These changes were unlikely to be cAMP dependent, as PGF2 alpha did not induce cAMP production, and the cAMP inducer forskolin was inhibitory. Moreover, phorbol ester treatment enhanced DNA synthesis to a greater extent in population 1 than in populations 3-5, and cotreatment with H-8 (at Km, approximately 10 microM) and staurosporine (at Km, approximately 0.01 microM) decreased the mitogenic effect of PGs in population 1, consistent with a reduction in protein kinase-C activation. These studies suggest that PGs activate less differentiated bone cells by a protein kinase-dependent event, whereas cAMP (induced by PGE1 and PGE2) decreases DNA and protein synthesis in more differentiated bone cells and tempers the increase in cellular activation found in population 1. Consequently, agents or events that increase the synthesis of specific PGs could differentially regulate, in positive and negative ways, biochemical activities in discrete bone cell populations.

Animals↗