Biomedical subjects

R T Franceschi

Publications and source records attributed to R T Franceschi.

At least 19 recordsLinked to original sources

PubMed2001

Cell-interactive alginate hydrogels for bone tissue engineering.

There is significant interest in the development of injectable carriers for cell transplantation to engineer bony tissues. In this study, we hypothesized that adhesion ligands covalently coupled to hydrogel carriers would allow one to control pre-osteoblast cell attachment, proliferation, and differentiation. Modification of alginate with an RGD-containing peptide promoted osteoblast adhesion and spreading, whereas minimal cell adhesion was observed on unmodified hydrogels. Raising the adhesion ligand density increased osteoblast proliferation, and a minimum ligand density (1.5-15 femtomoles/cm2) was needed to elicit this effect. MC3T3-E1 cells demonstrated increased osteoblast differentiation with the peptide-modified hydrogels, as confirmed by the up-regulation of bone-specific differentiation markers. Further, transplantation of primary rat calvarial osteoblasts revealed statistically significant increases of in vivo bone formation at 16 and 24 weeks with G4RGDY-modified alginate compared with unmodified alginate. These findings demonstrate that biomaterials may be designed to control bone development from transplanted cells.

3T3 Cells↗

PubMed2001

Matrix gamma-carboxyglutamic acid protein is a key regulator of PTH-mediated inhibition of mineralization in MC3T3-E1 osteoblast-like cells.

As part of its overall function as a major regulator of calcium homeostasis, PTH stimulates bone resorption and inhibits osteoblast-mediated biomineralization. To determine the basis for the inhibitory actions of this hormone, we compared the time course of PTH-dependent inhibition of mineralization in MC3T3-E1 osteoblast-like cells with changes in mRNA levels for several extracellular matrix proteins previously associated either with induction or inhibition of mineralization. Mineralizing activity was rapidly lost in PTH-treated cells ( approximately 30% inhibition after 3 h, 50% inhibition at 6 h). Of the proteins examined, changes in matrix gamma-carboxyglutamic acid protein were best correlated with PTH-dependent inhibition of mineralization. Matrix gamma-carboxyglutamic acid protein mRNA was rapidly induced 3 h after PTH treatment, with a 6- to 8-fold induction seen after 6 h. Local in vivo injection of PTH over the calvaria of mice also induced a 2-fold increase in matrix gamma-carboxyglutamic acid protein mRNA. Warfarin, an inhibitor of matrix gamma-carboxyglutamic acid protein gamma-carboxylation, reversed the effects of PTH on mineralization in MC3T3-E1 cells, whereas vitamin K enhanced PTH activity, as would be expected if a gamma-carboxyglutamic acid-containing protein were required for PTH activity. Levels of the other mRNAs examined were not well correlated with the observed changes in mineralization. Osteopontin, an in vitro inhibitor of mineralization, was induced approximately 4-fold 12 h after PTH addition. Bone sialoprotein mRNA, which encodes an extracellular matrix component most frequently associated with mineral induction, was inhibited by 50% after 12 h of PTH treatment. Osteocalcin mRNA, encoding the other known gamma-carboxyglutamic acid protein in bone, was also inhibited by PTH, but, again, with a significantly slower time course than was seen for mineral inhibition. Taken together, these results show that the rapid inhibition of osteoblast mineralization induced by in vitro PTH treatment is at least in part explained by induction of matrix gamma-carboxyglutamic acid protein.

1-Carboxyglutamic Acid↗

PubMed2000

Gene therapy for bone formation: in vitro and in vivo osteogenic activity of an adenovirus expressing BMP7.

Bone morphogenetic proteins (BMPs) are well-established agents for inducing orthotopic and ectopic bone formation. However, their clinical usefulness as regenerative agents may be limited by a short in vivo half-life and low specific activity. BMP gene therapy is an alternative route for exploiting the bone-inductive activity of this class of molecules. To test the feasibility of this approach, we examined the osteogenic activity of AdCMV-BMP7, an adenovirus containing BMP7 cDNA under control of the CMV promoter that was constructed using Cre/lox recombination (Hardy et al. [1997] J. Virol. 71:1842-1849). Adenovirus vectors were shown to readily infect a wide variety of cell types in vitro including osteoblasts, fibroblasts, and myoblasts. COS7 cells transduced with AdCMV-BMP7 produced high levels of BMP-7 (approximately 0.5 microg/10(6) cells). Furthermore, transduction of C2C12 murine myoblast cells with AdCMVBMP-7 suppressed the muscle phenotype and induced in vitro osteoblast differentiation. To test its in vivo biological activity, AdCMV-BMP7 was mixed with a bovine bone-derived collagen carrier (10(8) plaque-forming units virus/site) and was implanted into mouse muscle and dermal pouches. In both cases, an ossicle containing cortical and trabecular bone and a clearly defined marrow cavity formed at the site of virus implantation within 4 weeks. These data demonstrate that AdCMV-BMP7 transduced cells produce biologically active BMP-7 both in vitro and in vivo and show that gene therapy by direct viral transduction using a virus/matrix implant may be a viable route for stimulating bone regeneration.

Adenoviridae↗

PubMed2000

Gene therapy-directed osteogenesis: BMP-7-transduced human fibroblasts form bone in vivo.

An ex vivo gene therapy strategy was used to achieve localized skeletal regeneration in vivo. When an adenovirus vector engineered to express bone morphogenetic protein 7 transduced human gingival fibroblasts or rat dermal fibroblasts, these nonosteogenic tissues formed bone and supported the development of hematopoietic tissue when transplanted into immunocompromised mice. Transduced gingival fibroblasts formed marrow-containing ossicles in 100% of transplants after 1-2 weeks in vivo (n = 30). Immunostaining with murine and human-specific antisera raised against osteonectin and in situ hybridization of human-specific Alu genomic sequence demonstrated that the newly formed bone organ was a chimera of both the human donor and the mouse recipient cells. In experiments of greater clinical relevance, AdCMVBMP-7-transduced dermal fibroblasts repaired critical size skeletal defects in rat calvariae (n = 6). The results of this study suggest a bifunctional role of BMP-7-transduced fibroblasts. The transduced, nonosteogenic cells not only secreted biologically active BMP-7 in vitro and in vivo, but also differentiated into bone-forming cells in vivo. This model exploits the use of an easily biopsied, self-regenerating tissue such as gingiva or skin and suggests that local regeneration of tissues by ex vivo gene therapy may not require that autogenous cells be cultured from the tissue that is to be regenerated.

Adenoviridae↗

PubMed2000

Identification of a homeodomain binding element in the bone sialoprotein gene promoter that is required for its osteoblast-selective expression.

Bone sialoprotein is a 70-kDa extracellular matrix component that is intimately associated with biomineralization, yet the cis-acting elements of the Bsp gene that restrict its expression to mineralizing cells remain uncharacterized. To identify such elements, we analyzed a 2472-base pair fragment of the murine promoter that directs osteoblast-selective expression of a luciferase reporter gene and found that the region between -338 and -178 relative to the transcriptional start is crucial for its osteoblast-selective activity. We identified an element within this region that binds a protein complex in the nuclear extracts of osteoblastic cells and is required for its transcriptional activity. Introduction of a mutation that disrupts a homeodomain binding site within this sequence eliminates both its in vitro binding and nearly all of the osteoblastic-selective activity of the 2472-base pair promoter. We further found that the Dlx5 homeoprotein, which is able to regulate the osteoblast-specific osteocalcin promoter, can bind this element and stimulate its enhancer activity when overexpressed in COS7 cells. These data represent the first description of an osteoblast-specific element within the bone sialoprotein promoter and demonstrate its regulation by a member of a family of factors known to be involved in skeletogenesis.

Animals↗

PubMed2000

MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1.

The bone-specific transcription factor, Cbfa1, regulates expression of the osteocalcin (OCN) gene and is essential for bone formation. However, little is known about the mechanisms regulating Cbfa1 activity. This work examines the role of the MAPK pathway in regulating Cbfa1-dependent transcription. Stimulation of MAPK by transfecting a constitutively active form of MEK1, MEK(SP), into MC3T3-E1 preosteoblast cells increased endogenous OCN mRNA, while a dominant negative mutant, MEK(DN), was inhibitory. MEK(SP) also stimulated activity of a 147-base pair minimal OCN promoter, and this stimulation required an intact copy of OSE2, the DNA binding site for Cbfa1. Effects of MEK(SP) were specific to Cbfa1-positive osteoblast-like cells. A purified His-tagged Cbfa1 fusion protein was directly phosphorylated by activated recombinant MAPK in vitro. Furthermore, (32)P metabolic labeling studies demonstrated that MEK(SP) clearly enhanced phosphorylation of Cbfa1 in intact cells, while MEK(DN) decreased phosphorylation. The specific MEK1/MEK2 inhibitor, PD98059, inhibited extracellular matrix-dependent up-regulation of the OCN promoter, indicating that the MAPK pathway and, presumably, Cbfa1 phosphorylation are also required for responsiveness of osteoblasts to extracellular matrix signals. This study is the first demonstration that Cbfa1 is controlled by MAPKs and suggests that this pathway has an important role in the control of osteoblast-specific gene expression.

Animals↗

PubMed2000

Engineered bone development from a pre-osteoblast cell line on three-dimensional scaffolds.

Bone regeneration is based on the hypothesis that healthy progenitor cells, either recruited or delivered to an injured site, can ultimately regenerate lost or damaged tissue. Three-dimensional porous polymer scaffolds may enhance bone regeneration by creating and maintaining a space that facilitates progenitor cell migration, proliferation, and differentiation. As an initial step to test this possibility, osteogenic cells were cultured on scaffolds fabricated from biodegradable polymers, and bone development on these scaffolds was evaluated. Porous polymer scaffolds were fabricated from biodegradable polymers of lactide and glycolide. MC3T3-E1 cells were statically seeded onto the polymer scaffolds and cultured in vitro in the presence of ascorbic acid and beta-glycerol phosphate. The cells proliferated during the first 4 weeks in culture and formed a space-filling tissue. Collagen messenger RNA levels remained high in these cells throughout the time in culture, which is consistent with an observed increase in collagen deposition on the polymer scaffold. Mineralization of the deposited collagen was initially observed at 4 weeks and subsequently increased. The onset of mineralization corresponded to increased mRNA levels for two osteoblast-specific genes: osteocalcin and bone sialoprotein. Culture of cell/polymer constructs for 12 weeks led to formation of a three-dimensional tissue with architecture similar to that of native bone. These studies demonstrate that osteoblasts within a three-dimensional engineered tissue follow the classic differentiation pathway described for two-dimensional culture. Polymer scaffolds such as these may ultimately be used clinically to enhance bone regeneration by delivering or recruiting progenitor cells to the wound site.

3T3 Cells↗

PubMed2000

Parathyroid hormone-related protein down-regulates bone sialoprotein gene expression in cementoblasts: role of the protein kinase A pathway.

PTH-related protein (PTHrP) acts as a paracrine and/or autocrine regulator of cell proliferation, apoptosis, and differentiation and is implicated in tooth development. The current studies employed cementoblasts to determine the role(s) and mechanisms of PTHrP in regulating cementum formation. Results demonstrated that PTHrP repressed gene expression and protein synthesis of bone sialoprotein (BSP) and abolished cementoblast-mediated biomineralization in vitro. The BSP gene inhibition required protein synthesis. The PTHrP analog (1-31) and other activators of the PKA pathway (3-isobutyl-1-methylxathine (IBMX), forskolin (FSK) and Sp-Adenosine-3', 5'-cyclic monophosphorothioate (Sp-cAMPss) also down-regulated BSP gene expression and blocked cementoblast-mediated biomineralization. In contrast, the PTHrP analog (7-34), a PTHrP antagonist, and the activators of the PKC pathway [phorbol 12-myristate 13-acetate (PMA) and phorbol 12, 13-dibutyrate (PDBu)] promoted BSP gene expression. In addition, the PKA pathway inhibitor (9-(2-tetrahydrofuryl) adenine (THFA) partially, but significantly reversed the PTHrP-mediated down-regulation of BSP gene expression. Furthermore, THFA alone significantly increased BSP messenger RNA (mRNA) expression in cementoblasts. In contrast, the inhibitor of the PKC pathway (GF109203X) did not reverse the PTHrP inhibitory effect on BSP gene expression. Furthermore, GF109203X alone dramatically reduced the BSP transcript levels. These data indicate that the cAMP/PKA pathway mediates the PTHrP-mediated down-regulation of BSP mRNA expression in cementoblasts; and furthermore, this pathway may, through an intrinsic inhibition mechanism, regulate the basal level of BSP mRNA expression. In contrast, the activation of PKC promotes BSP gene expression. These data provide new insights into the molecular mechanisms involved in PTHrP regulation of cementogenesis.

1-Methyl-3-isobutylxanthine↗

PubMed1999

The developmental control of osteoblast-specific gene expression: role of specific transcription factors and the extracellular matrix environment.

Bone formation is a carefully controlled developmental process involving morphogen-mediated patterning signals that define areas of initial mesenchyme condensation followed by induction of cell-specific differentiation programs to produce chondrocytes and osteoblasts. Positional information is conveyed via gradients of molecules, such as Sonic Hedgehog that are released from cells within a particular morphogenic field together with region-specific patterns of hox gene expression. These, in turn, regulate the localized production of bone morphogenetic proteins and related molecules which initiate chondrocyte- and osteoblast-specific differentiation programs. Differentiation requires the initial commitment of mesenchymal stem cells to a given lineage, followed by induction of tissue-specific patterns of gene expression. Considerable information about the control of osteoblast-specific gene expression has come from analysis of the promoter regions of genes encoding proteins like osteocalcin that are selectively expressed in bone. Both general and tissue-specific transcription factors control this promoter. Osf2/Cbfa1, the first osteoblast-specific transcription factor to be identified, is expressed early in the osteoblast lineage and interacts with specific DNA sequences in the osteocalcin promoter essential for its selective expression in osteoblasts. The OSF2/CBFA1 gene is necessary for the development of mineralized tissues, and its mutation causes the human disease, cleidocranial dysplasia. Committed osteoprogenitor cells already expressing Osf2/Cbfa1 must synthesize a collagenous ECM before they will differentiate. A cell:ECM interaction mediated by integrin-type cell-surface receptors is essential for the induction of osteocalcin and other osteoblast-related proteins. This interaction stimulates the binding of Osf2/Cbfa1 to the osteocalcin promoter through an as-yet-undefined mechanism.

Animals↗

PubMed1999

Cloning of a 2.5 kb murine bone sialoprotein promoter fragment and functional analysis of putative Osf2 binding sites.

Bone sialoprotein (BSP) is an extracellular matrix protein that is intimately associated with the process of biomineralization. Osf2, a member of the Cbf/runt family of transcription factors, is required for the development of osteoblasts in vivo and has been reported to stimulate the transcription of BSP when overexpressed in mesenchymal cell lines. To investigate the role of Osf2 in BSP expression, we cloned a 2.5 kb fragment of a 5' untranscribed sequence from the murine BSP gene and evaluated it for putative Osf2 binding sites. This promoter, which was able to direct 5- to 10-fold higher levels of luciferase reporter expression in osteoblastic cells than in nonbone cell lines, contains two consensus core binding sites for members of the Cbf/runt family. One, at -61 relative to the start of transcription, is within a region having 75% overall sequence identity with the rat and human BSP promoters. The other is located at -1335, outside this highly conserved region. Neither site is completely conserved in the rat or human sequences. Only the -1335 site was able to bind a protein in nuclear extracts of osteoblastic cells, and this protein was identified as Osf2. Despite this in vitro binding ability, we detected no significant enhancer activity in the -1335 element when placed in front of a minimal osteocalcin promoter driving a luciferase reporter gene in osteoblastic cells nor any loss in transcriptional activity of a 5' promoter deletion which eliminated this element as compared with the full-length 2.5 kb promoter. These results suggest that Osf2 binding to the BSP promoter is not essential for its osteoblast-selective expression.

3T3 Cells↗

PubMed1999

Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential.

A series of subclonal cell lines with high or low differentiation/mineralization potential after growth in the presence of ascorbic acid (AA) were derived from murine MC3T3-E1 cells. Subclones were characterized in terms of their ability to mineralize a collagenous extracellular matrix both in vitro and in vivo and express osteoblast-related genes. When compared with nonmineralizing cells, mineralizing subclones selectively expressed mRNAs for the osteoblast markers, bone sialoprotein (BSP), osteocalcin (OCN), and the parathyroid hormone (PTH)/parathyroid hormone-related protein (PTHrP) receptor. In contrast, alkaline phosphatase mRNA was present in certain nonmineralizing as well as mineralizing subclones, suggesting that its expression may be subject to different controls from other osteoblast markers. Only highly differentiating subclones exhibited strong AA-dependent induction of a transiently transfected OCN promoter-luciferase reporter gene, indicating that there was a good correlation between mRNA levels and transcriptional activity. Consistent with its postulated role in biomineralization, BSP as measured by Western blotting was only present in mineralizing subclones. After implantation into immunodeficient mice, highly differentiating subclones formed bone-like ossicles resembling woven bone, while poorly differentiating cells only produced fibrous tissue. Interestingly, subclones with both high and low differentiation potential produced similar amounts of collagen in culture and expressed comparable basal levels of mRNA encoding Osf2/Cbfa1, an osteoblast-related transcription factor. Although some strongly differentiating cells exhibited a modest AA-dependent up-regulation of Osf2/Cbfa1 mRNA, there was no clear relationship between levels of this message and induction of mRNAs for other differentiation markers. Thus, the mere presence of Osf2/Cbfa1 in a subclone was not sufficient for osteoblast differentiation. These subclones will be very useful for studying critical events in osteoblast differentiation and mineralization.

Animals↗

PubMed1998

Role of the alpha2-integrin in osteoblast-specific gene expression and activation of the Osf2 transcription factor.

Extracellular matrix molecules such as type I collagen are required for the adhesion, migration, proliferation, and differentiation of a number of cell types including osteoblasts. Matrix components often affect cell function by interacting with members of the integrin family of cell surface receptors. Previous work showed that collagen matrix synthesis, induced by addition of ascorbic acid to cells, precedes and is essential for the expression of osteoblast markers and induction of the osteocalcin promoter in murine MC3T3-E1 cells. This later response requires OSE2, the promoter element recognized by Osf2 (also called Cbfa1/AML3/PEBP2alphaA), a recently identified osteoblast-specific transcription factor. Osteoblasts express several integrins including alpha2beta1 which is a major receptor for type I collagen. This paper examines the role of the alpha2-integrin subunit in osteocalcin promoter activation and osteoblast differentiation. Disruption of alpha2-integrin-ECM interactions with a blocking antibody or DGEA peptide containing the cell-binding domain of type I collagen blocked activation of the mouse osteocalcin gene 2 promoter by ascorbic acid as well as induction of endogenous osteocalcin mRNA and mineralization. Furthermore, anti-alpha2-integrin blocking antibody or peptide reduced ascorbic acid-dependent binding of Osf2 to OSE2 without affecting levels of transcription factor mRNA. Time course studies revealed that ascorbic acid-dependent binding of Osf2 to OSE2 preceded increases in osteocalcin and bone sialoprotein expression and this increase in Osf2 binding was not accompanied by comparable changes in levels of transcription factor mRNA or protein. Taken together, these studies demonstrate that an alpha2-integrin-collagen interaction is required for activation of Osf2 and induction of osteoblast-specific gene expression. Furthermore, matrix signals may regulate Osf2 through a post-translational pathway or via an accessory factor.

3T3 Cells↗

PubMed1998

Functional hierarchy between two OSE2 elements in the control of osteocalcin gene expression in vivo.

Osteocalcin gene expression is initiated perinatally and is restricted to mature osteoblasts and odontoblasts. Because their pattern of expression is highly restricted, the osteocalcin genes are excellent tools to study osteoblast-specific gene expression. To define the mechanisms of osteocalcin cell-specific gene expression in vivo, we generated transgenic mice harboring deletion mutants of the promoter region of OG2, one of the mouse osteocalcin genes. We show here that only 647 base pairs of this promoter are sufficient to confer cell-specific and time-specific expression to a reporter gene in vivo. This promoter fragment contains two copies of OSE2. This osteoblast-specific cis-acting element binds Osf2, a recently characterized osteoblast-specific transcription factor (Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L., and Karsenty, G. (1997) Cell 89, 747-754). We also demonstrate that the proximal OSE2 element is critical to confer an osteoblast-specific, developmentally regulated pattern of expression to a reporter gene. The other OSE2 element, located more upstream and presenting a lower affinity for Osf2, affects only weakly OG2 promoter activity. These data demonstrate the crucial role of Osf2 in controlling osteocalcin gene expression. Since osteocalcin synthesis is a hallmark of the differentiated osteoblast phenotype, these results suggest that, beyond its developmental function, Osf2 is also required for the maintenance of the osteoblast phenotype postnatally.

Animals↗

PubMed1998

Glucocorticoid stimulation of Na+-dependent ascorbic acid transport in osteoblast-like cells.

Ascorbic acid (AA) is an essential cofactor for osteoblast differentiation both in vivo and in vitro. Before it can function, this vitamin must be transported into cells via a specific Na+-dependent AA transporter. In this study, we examine the regulation of this transport activity by glucocorticoids, a class of steroid hormones known to stimulate in vitro osteoblast differentiation. Dexamethasone stimulated Na+-dependent AA transport activity approximately twofold in primary rat calvarial osteoblasts. Effects of hormone on ascorbic acid transport were rapid (detected within 24 h) and were maximally stimulated by 25-50 nM dexamethasone. Similar effects of dexamethasone on transport activity were also observed in murine MC3T3-E1 cells. This preosteoblast cell line was used for a more detailed characterization of the glucocorticoid response. Transport activity was stimulated selectively by glucocorticoids (dexamethasone > corticosterone) relative to other steroid hormones (progesterone and 17-beta-estradiol) and was blocked when cells were cultured in the presence of cycloheximide, a protein synthesis inhibitor. Kinetic analysis of AA transporter activity in control and dexamethasone-treated cells indicated a Km of approximately 17 microM for both groups. In contrast, dexamethasone increased Vmax by approximately 2.5-fold. Cells also contained an Na+-independent glucose transport activity that has been reported in other systems to transport vitamin C as oxidized dehydroascorbic acid. In marked contrast to Na+-dependent AA transport, this activity was inhibited by dexamethasone. Thus, glucocorticoids increase Na+-dependent AA transport in osteoblasts, possibly via up-regulation of transporter synthesis, and this response can be resolved from actions of glucocorticoids on glucose transport.

Animals↗

PubMed1997

Ascorbic acid-dependent activation of the osteocalcin promoter in MC3T3-E1 preosteoblasts: requirement for collagen matrix synthesis and the presence of an intact OSE2 sequence.

Osteocalcin is a hormonally regulated calcium-binding protein made almost exclusively by osteoblasts. In normal cells, osteocalcin expression requires ascorbic acid (AA), an essential cofactor for osteoblast differentiation both in vivo and in vitro. To determine the mechanism of this regulation, subclones of MC3T3-E1 preosteoblasts were transiently transfected with 1.3 kb of the mouse osteocalcin gene 2 promoter driving expression of firefly luciferase. AA stimulated luciferase activity 20-fold after 4-5 days. This response was stereospecific to L-ascorbic acid and was only detected in MC3T3-E1 subclones showing strong AA induction of the endogenous osteocalcin gene. Similar results were also obtained in MC3T3-E1 cells stably transfected with the osteocalcin promoter. A specific inhibitor of collagen synthesis, 3,4-dehydroproline, blocked AA-dependent induction of promoter activity, indicating that regulation of the osteocalcin gene requires collagen matrix synthesis. Deletion analysis of the mOG2 promoter identified an essential region for AA responsiveness between -147 and -116 bp. This region contains a single copy of the previously described osteoblast-specific element, OSE2. Deletion and mutation of OSE2 in DNA transfection assays established the requirement for this element in the AA response. Furthermore, DNA-binding assays revealed that MC3T3-E1 cells contain OSF2, the nuclear factor binding to OSE2, and that binding of OSF2 to OSE2 is up-regulated by AA treatment. Taken collectively, our results indicate that an intact OSE2 sequence is required for the induction of osteocalcin expression by AA.

Animals↗

PubMed1996

PTH/PTHrP receptor is temporally regulated during osteoblast differentiation and is associated with collagen synthesis.

The temporal sequence of PTH/PTHrP receptor mRNA, binding, biologic activity, and its dependence on matrix synthesis was determined using MC3T3-E1 preosteoblast-like cells and primary rat calvarial cells in vitro. Osteoblastic cells were induced to differentiate and form mineralized nodules with the addition of ascorbic acid and beta-glycerophosphate, and samples were collected from 0-26 days of culture. DNA levels as determined by fluorometric analysis increased 12- and 17-fold during the collection period for both MC3T3-E1 and primary calvarial cells respectively. Steady state mRNA levels for the PTH/PTHrP receptor as determined by northern blot analysis, were initially low for both cell types, peaked at day 4 and 5 for MC3T3-E1 and primary calvarial cells respectively, and declined thereafter. Competition binding curves were performed during differentiation using 125I-PTHrP. The numbers of receptors per microgram DNA were greatest at days 3 and 5 for MC3T3-E1 and primary calvarial cells respectively. The biologic activity of the receptor was evaluated by stimulating the cells with 10 nM PTHrP and determining cAMP levels via a binding protein assay. The PTHrP-stimulated cAMP levels increased 5-fold to peak values at day 5 for MC3T3-E1! cells and 6-fold to peak values at day 4 for the primary calvarial cells. Ascorbic acid was required for maximal development of a PTH-dependent cAMP response since ascorbic acid-treated MC3T3-E1 cells had twice the PTH-stimulated cAMP levels as non-treated cells. When the collagen synthesis inhibitor 3,4-dehydroproline was administered to MC3T3-E1 cultures prior to differentiation, there was a subsequent diminution of the PTH/PTHrP receptor mRNA gene expression and numbers of receptors per cell; however, if administered after the initiation of matrix synthesis there was no reduction in PTH/PTHrP receptor mRNA. These findings indicate that the PTH/PTHrP receptor is associated temporally at the level of mRNA, protein, and biologic activity, with a differentiating, matrix-producing osteoblastic cell in vitro.

3T3 Cells↗

PubMed1995

Effects of differentiation and transforming growth factor beta 1 on PTH/PTHrP receptor mRNA levels in MC3T3-E1 cells.

TGF beta has opposing effects on osteoblasts which are thought to be differentiation stage dependent; however, little is known concerning the effects of TGF beta on osteoblastic characteristics at different stages of maturation. The purpose of this study was to characterize the pattern of mRNA expression for the PTH/PTHrP receptor during normal osteoblastic differentiation in vitro, and evaluate the effects of TGF beta 1 on PTH/PTHrP receptor and osteocalcin (OCN) steady-state mRNA at different stages of osteoblastic differentiation. MC3T3-E1 preosteoblasts were plated at low density and induced to differentiate with ascorbic acid and beta-glycerophosphate. The first group served as a vehicle control and the remaining five groups received a single 48 h TGF beta 1 (3.0 ng/ml)-pulse staggered on a weekly basis for 30 days. Cell cultures were harvested weekly and evaluated for: steady-state PTH/PTHrP receptor and OCN mRNA levels via northern analysis, calcium and phosphorous levels, bone nodules via Von Kossa staining, alkaline phosphatase enzyme levels, and hydroxyproline levels. Group 1 (control) samples followed a normal pattern of proliferation, extracellular matrix deposition, and mineralization. PTH/PTHrP receptor and OCN mRNA expression increased 8-fold and 10-fold respectively, over the collection periods. When TGF beta 1 was administered during the first 48 h period (group 2) while cells were rapidly proliferating, there was a persistent inhibition of PTH/PTHrP receptor expression and a striking reduction in OCN mRNA expression at all time points. There was also a down-regulation of PTH/PTHrP receptor and OCN expression when TGF beta 1 was administered later during osteoblast differentiation (groups 3-6); however, these effects were not persistent. In addition there was a total lack of bone nodule formation in group two cultures, whereas groups 3-6 had increasing bone nodule formation because the TGF beta 1 was administered later in the culture period. These studies indicate that expression of the PTH/PTHrP receptor increases with osteoblastic differentiation and suggest that TGF beta 1 inhibits osteoblastic maturation with more persistent effects found in less differentiated osteoblastic cells.

3T3 Cells↗

PubMed1995

Mineralization of bone-like extracellular matrix in the absence of functional osteoblasts.

When grown in medium containing ascorbic acid and beta-glycerol phosphate, mouse MC3T3-E1 cells express an osteoblast phenotype and produce a highly mineralized extracellular matrix. The purpose of this study was to independently examine the role of the collagenous matrix and functional osteoblasts on the mineralization process. Cultures with and without an extensive collagenous matrix were prepared by growing MC3T3-E1 cells in the presence and absence of ascorbic acid. Matrix-rich cultures mineralized at much lower calcium phosphate ion products than nonmatrix cultures. At higher ion products, spontaneous precipitation in the medium and cell layers of nonmatrix cultures were observed. In contrast, mineral in matrix-rich cultures was still exclusively associated with collagen fibrils and not with ectopic sites in the cell layer or medium. To examine the effect of cell viability on matrix mineralization, cells were grown 8 or 16 days in the presence of ascorbic acid, then killed and incubated in a mineralizing medium. Significant mineralization was not observed in the collagenous matrix of 8-day killed cultures or age-matched controls. At 16 days mineral was associated with collagen fibrils at specific foci in the matrix of both viable and killed cultures. This observation is consistent with the concept that collagenous matrices must undergo a maturation process before they can support a mineral induction and growth. It further shows that osteoblast-like cells are not required for mineralization of mature matrices, but are required for matrix maturation.

3T3 Cells↗