Biomedical subjects

J E Aubin

Publications and source records attributed to J E Aubin.

At least 19 recordsLinked to original sources

PubMed1998

LIF, but not IL-6, regulates osteoprogenitor differentiation in rat calvaria cell cultures: modulation by dexamethasone.

Cytokines of the interleukin 6 (IL-6) subfamily are a group of factors produced by osteoblasts and acting through the same transducing element, membrane protein gp130. We have previously shown that exogenous (added to the culture medium) leukemia inhibitory factor (LIF) inhibits bone nodule formation and expression of osteoblast-associated genes in fetal rat calvaria (RC) cell cultures and that dexamethasone (Dex) increases the ID50 of LIF. To investigate the respective roles of IL-6-related cytokines and receptors in osteprogenitor differentiation, and their regulatory interplay with Dex, we used reverse transcribed polymerase chain reaction, bioassay, and blocking antibody techniques to assess the time courses of LIF, IL-6, LIF transmembrane receptor, IL-6 receptor, and gp130 expression in RC cell cultures grown with and without Dex. The levels of the mRNAs for IL-6, LIF, and gp130 decreased concomitantly with the formation of bone nodules. Dex treatment, which stimulates bone nodule formation, reduced the expression of LIF and IL-6 mRNAs and IL-6 bioactivity in the culture medium. LIF treatment strongly stimulated the expression of IL-6. Incubation with anti-LIF antibodies increased the number of nodules, while an antibody blocking IL-6 activity had little or no effect on nodule numbers and did not antagonize the action of exogenous LIF, indicating that IL-6 does not mediate the action of LIF in this system. Moreover, although exogenously added IL-6 was active in the cultures as noted by a reduction of nodule mineralization, it had no effect on nodule numbers, i.e., on osteoprogenitor differentiation, in the presence or absence of Dex. In conclusion, IL-6, LIF, and their receptors are expressed throughout the time-course of osteogenesis in RC cell cultures. However, only LIF, but not IL-6, appears to play a significant role in autocrine regulation of osteoblastic differentiation in this system. The antagonist action of Dex on the effects of exogenously added LIF, as well as the bone-promoting action of Dex in RC cell cultures, could be exerted partly through the down-regulation of the expression of endogenous LIF.

Animals↗

PubMed1997

The mature osteoblast phenotype is characterized by extensive plasticity.

While both morphological and biochemical-molecular attributes demarcate differentiation stages in specific cell and tissue types, what constitutes necessary and sufficient expression to define particular cell types is not always known. For example, mature osteoblasts (OBs) are defined morphologically as the cuboidal, biosynthetically active, basophilic cells residing on bone surfaces and responsible for the deposition of osteoid matrix. However, several recent observations suggest that not all mature OBs are identical. To explore further the validity of the hypothesis that heterogeneity of phenotype exists among mature OBs, we grew fetal rat calvaria cells in vitro at low density under conditions in which bone nodules form and mineralize in isolation of other contaminating cell and colony types. Cells resident in mature OB colonies, i.e., those comprising mainly cuboidal cells associated with an osteoid matrix that had begun to mineralize, were analyzed in situ for protein expression by immunocytochemistry with antibodies against collagen type I, alkaline phosphatase, osteopontin, bone sialoprotein, and osteocalcin. Consistent with the expected phenotype of mature OBs, many OBs expressed high levels of all of these markers, but strikingly even adjacent morphologically indistinguishable cuboidal OBs had differences in protein expression, especially in relation to osteopontin, bone sialoprotein, and osteocalcin expression. Double-labeling with Hoechst 33258 and osteocalcin indicated that the variation in antibody labeling intensity/protein expression appeared independent of a variation in cell cycle. To further ascertain the extent of this heterogeneity, 20 single cells were micromanipulated from colonies and subjected to poly(A)-PCR to analyze the simultaneous coexpression profiles of the same five markers analyzed by immunocytochemistry and two other markers, the OB-osteocyte transition marker E11 and the parathyroid hormone/parathyroid hormone-related protein receptor. Notably, the repertoire of genes expressed and their levels of expression varied markedly in individual OBs. The observed heterogeneity suggests that the mature OB phenotype is not a single unique phenotype but rather encompasses a flexible pattern of expression from the repertoire of OB-associated markers.

Alkaline Phosphatase↗

PubMed1997

Cell sorting enriches osteogenic populations in rat bone marrow stromal cell cultures.

The presence of multiple cell types in bone marrow stromal populations complicates interpretation of cytokine and hormone effects on the osteoprogenitors present, indicating a need for a method for purification of the osteoprogenitor population. Flow cytometric sorting of 7 day primary rat bone marrow stromal cell cultures was performed on the basis of alkaline phosphatase (AP) expression with an antibody against AP (RBM 211.13). The resultant AP(high), AP(low), or control cells were plated to determine osteoprogenitor, macrophage, and adipocyte distribution and frequency. Approximately 50% of osteoprogenitor/bone nodule-forming cells were lost during processing/sorting when compared with unsorted controls. Nevertheless, within the AP(high) fraction, the numbers of AP-positive colonies and osteoprogenitors (bone nodules) were significantly enriched compared with the unfractionated control; the increase in osteoprogenitor frequency ranged from approximately 2 to 100-fold. There were few assayable osteoprogenitors in either the AP(high) or AP(low) fractions in the absence of dexamethasone (dex), suggesting that RBM stroma contains largely dex-dependent osteoprogenitor populations, and that dex may regulate osteoprogenitors subsequent to the upregulation of AP. Osteoprogenitor/bone nodule numbers in either the AP(high) or AP(low) fraction did not follow a linear relationship with decreasing plating density. The AP(high) fraction of cells was depleted for adipocyte and macrophage colonies. In contrast, within the AP(low) fraction of cells, adipocyte and macrophage colonies were consistently enriched. We conclude that flow-cytometric sorting of RBM stromal populations according to high or low AP expression is an effective technique for enrichment of AP-positive colonies and osteoprogenitors/bone nodule-forming cells.

Adipocytes↗

PubMed1997

Osteogenic protein-1 up-regulation of the collagen X promoter activity is mediated by a MEF-2-like sequence and requires an adjacent AP-1 sequence.

Bone morphogenetic proteins induce chondrogenesis and osteogenesis in vivo. To investigate molecular mechanisms involved in chondrocyte induction, we examined the effect of osteogenic protein (OP)-1/bone morphogenetic protein-7 on the collagen X promoter. In rat calvaria-derived chondrogenic C5.18 cells, OP-1 up-regulates collagen X mRNA levels and its promoter activity in a cell type- specific manner. Deletion analysis localizes the OP-1 response region to 33 bp (-310/-278), which confers OP-1 responsiveness to both the minimal homologous and heterologous Rous sarcoma virus promoter. Transforming growth factor-beta2 or activin, which up-regulates the expression of a transforming growth factor-beta-inducible p3TP-Lux construct, has little effect on collagen X mRNA and on this 33-bp region. Mutational analysis shows that both an AP-1 like sequence (-294/-285, TGAATCATCA) and an A/T-rich myocyte enhancer factor (MEF)-2 like sequence (-310/-298, TTAAAAATAAAAA) in the 33-bp region are necessary for the OP-1 effect. Gel shift assays show interaction of distinct nuclear proteins from C5.18 cells with the AP-1-like and the MEF-2-like sequences. OP-1 rapidly induces nuclear protein interaction with the MEF-2-like sequence but not with the AP-1 like sequence. MEF-2-like binding activity induced by OP-1 is distinct from the MEF-2 family proteins present in C2C12 myoblasts, in which OP-1 does not induce collagen X mRNA or up-regulate its promoter activity. In conclusion, we identified a specific response region for OP-1 in the mouse collagen X promoter. Mutational and gel shift analyses suggest that OP-1 induces nuclear protein interaction with an A/T-rich MEF-2 like sequence, distinct from the MEF-2 present in myoblasts, and up-regulates collagen X promoter activity, which also requires an AP-1 like sequence.

Activins↗

PubMed1997

The ERR-1 orphan receptor is a transcriptional activator expressed during bone development.

We studied the expression of estrogen-related receptor ERR-1 during mouse embryonic development. ERR-1 mRNA is present in bones formed by both the endochondral and intramembranous routes, and the onset of its expression coincides with bone formation. By RT-PCR experiments, we found that ERR-1, but not the related receptor ERR-2, is expressed in osteoblastic osteosarcoma cell lines as well as in primary osteoblastic cell populations derived from normal human bone. By gel shift analysis we found that ERR-1 binds as a monomer specifically to the SFRE sequence (SF-1-responsive-element; TCAAGGTCA). Mutation analysis revealed that both the core AGGTCA motif and the TCA 5'-extension are required for efficient ERR-1 binding. In transient transfection assays, ERR-1 acts as a potent transactivator through the SFRE sequence. This effect is cell-specific since ERR-1 activates transcription in the rat osteosarcoma cell line ROS 17.2/8 as well as in HeLa, NB-E, and FREJ4 cells but not in COS1 and HepG2 cells. Notably, the osteopontin (a protein expressed by osteoblasts and released in the bone matrix) gene promoter is a target for ERR-1 transcriptional regulation. Our findings suggest a role for ERR-1 in bone development and metabolism.

Animals↗

PubMed1997

Characterization of a monoclonal antibody recognizing mast cells.

Immunohistochemical screening for monoclonal antibodies prepared by immunization of mice with a rat osteoblastic cell population led to identification of one antibody that reacted against a small population of cells present in the soft connective tissue compartment of 21 days fetal rat calvaria. The morphology of the cells and the immunohistochemical staining characteristics (a distinct intracellular granular pattern) suggested that the antibody might be reacting specifically against mast cells. We used combined histochemistry and immunohistochemistry to further characterize this antibody, designated RCJ102. Cryosections containing calvaria bone, soft connective tissues and skin were prepared from the top of the head of 21 days fetal rats, and from adult rats cryosections of lung, muscle, adipose tissue and small intestine were prepared. Some sections were labelled by indirect immunofluorescence with RCJ102; corresponding sections were labelled histochemically with toluidine blue. There was a direct correspondence between mast cells identified histochemically and cells labelling with RCJ102 in all tissues except intestine, in which the mast cell detectable by histochemistry were not labelled by RCJ102. These results suggest that the RCJ102 antibody will be a valuable new reagent for further elucidation of the heterogeneity described between connective tissue and intestinal mucosal mast cells.

Animals↗

PubMed1996

CD44 expression in fetal rat bone: in vivo and in vitro analysis.

Knowledge of the expression pattern of the cell surface glycoprotein CD44 in the development and differentiation of bone is limited. We investigated CD44 expression (a) in bone sections of 21-day-old fetal rat calvaria (RC), metatarsals, and tibiae, (b) in primary cultures of RC cells undergoing differentiation in vitro, and (c) in three rat osteosarcoma cell lines: ROS 17/2.8, UMR 106.01, and UMR 106.06. By immunocytochemistry, Western, Northern and reverse transcription polymerase chain reaction analyses, we found that osteoblastic cells express the 'hematopoietic' or 'standard' CD44 (CD44s) isoform. Osteoblastic cells in vivo and in vitro stained at all detectable stages of differentiation, but intercellular heterogeneity of CD44s staining was evident, with lesser staining in preosteoblastic cells and greater staining in mature osteoblasts and osteocytes. As cells in RC cultures differentiated and formed bone in vitro, CD44s mRNA and protein levels as measured on immunoblots were invariant. All three osteosarcoma cell lines expressed CD44s mRNA and protein. The synthetic glucocorticoid dexamethasone, which stimulates osteogenesis in RC cells in vitro and regulates a number of osteoblast-associated genes, had no apparent effect on either CD44s protein or mRNA levels. The widespread presence of CD44s in osteoblastic cells at various maturational stages suggests that further analyses will be required to determine what role CD44s may play in osteogenesis and in bone tissue organization.

Animals↗

PubMed1996

Monoclonal antibodies as tools for studying the osteoblast lineage.

Knowledge of the number and kinds of differentiation steps characterizing cells of the osteoblast lineage is inadequate. To analyze further osteoblast differentiation, a number of labs have generated monoclonal antibodies to osteogenic cells, derived from both normal bone and osteosarcomas. A variety of immunolabelling patterns on primary cell cultures, cell lines, and tissue sections has been reported, including cell surface, cytoplasmic, and extracellular matrix-associated patterns. Most of the antibodies selected recognize predominantly the mature osteoblast and osteocyte; in addition, however, antibodies have been generated that recognize pre-osteoblasts. Some recognize cells of both the osteoblast and chondroblast lineages and may contribute to a better understanding of the lineage and phenotypic relationships between these two cell types. In addition to recognition in vivo of cell subpopulations of discrete maturational stages, changes in the immunolabelling patterns in vitro have also documented a differentiation sequence in cells undergoing osteogenesis in cell and tissue cultures. In at least two cases, the antibodies have been used to isolate subpopulations of cells from bone, including relatively pure populations of osteocytes. With the exception of several antibodies that are against alkaline phosphatase or known matrix proteins including osteocalcin, the nature of the macromolecular species recognized by most of the antibodies generated to date are unknown. Recently, however, one antibody was used to clone the cDNA for the beta-galactoside-binding lectin, galectin 3 or epsilon binding protein (epsilon BP; IgE-binding protein; Mac-2), from a lambda gt11 osteoblast expression library; another was used to clone from an ROS 17/2.8-COS cell expression library the cDNA for OTS-8, a putative target gene of early response genes stimulated in response to phorbol esters in MC3T3-E1 cells. Neither of these macromolecules had previously been identified in bone cells, but the recent molecular and cellular analyses have shown them to be developmentally and/or hormonally regulated in osteoblastic cells. These antibodies extend the available markers and support earlier observations that a variety of molecules are differentially expressed by cells at different stages of the osteoblast lineage. This chapter will not be an exhaustive survey of all immunocytochemical and immunohistochemical analyses of osteogenic cells and tissues but will focus on the approach of eliciting novel monoclonal antibodies by the injection of osteogenic cells or crude bone extracts and its potential for establishing new markers of the osteoblast lineage. We have not included a large number of studies documenting the use of antibodies raised against several known bone matrix proteins; while these have been crucial in developing our current understanding of osteogenic differentiation, we sought rather to highlight the potential of the "random" injection approach.

Animals↗

PubMed1996

Expression and regulation of galectin 3 in rat osteoblastic cells.

Galectin 3 is an endogenous soluble beta-galactoside-specific lectin originally identified and termed epsilon BP or IgE-binding protein in rat basophilic leukemia cells, but its wide tissue distribution and the multiple contexts in which it has been isolated have suggested that its function may not be limited to IgE binding but may include a role in cell growth regulation and differentiation, neoplastic transformation, and cell adhesion (Liu, 1990, Crit. Rev. Immunol., 10:289-306; Barondes et al., 1994, J. Biol. Chem., 269:20807-20810). After immunoscreening of a lambda gt11 cDNA expression library made from bone-nodule forming cultures of fetal rat calvaria (RC) cells with an antibody raised against osteoblastic cells (Turksen et al., 1992, J. Histochem. Cytochem., 40:1339-1352), three cDNA clones were isolated and sequenced; the sequence matched that of rat galectin 3. Galectin 3 mRNA was detected in various fetal and adult rat tissues, including calvaria and cultured RC cells. In RC cells and the rat osteosarcoma cell line ROS 17/2.8, galectin 3 mRNA expression increased with time in culture, in contrast to its behavior in fetal rat skin fibroblasts (RSF) in which its expression decreased with time in culture. In a second rat osteosarcoma line, UMR 106.01, galectin 3 mRNA was almost nondetectable. The synthetic glucocorticoid dexamethasone (Dex) enhanced galectin 3 expression in RSF cell cultures, while 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) had no significant effect. In contrast, Dex downregulated and 1,25(OH)2D3 upregulated galectin 3 expression in RC and ROS 17/2.8 cells, especially at later time points in culture when expression of osteoblast-associated differentiation markers by these cell types is most marked. Immunolabeling with an antibody against rat galectin 3 to identify galectin 3 protein showed that cells labelled within both the ROS 17/2.8 and RC populations but with marked intercellular heterogeneity of intensity. Our data support the conclusion that galectin 3 is a previously unrecognized product of osteoblastic cells, that galectin 3 mRNA and protein expression increases with time in vitro concomitant with other markers of osteogenesis, including formation of bone nodules and expression of osteoblast-associated markers such as alkaline phosphatase, bone sialo-protein, and osteocalcin, and that its expression is regulated by hormones such as glucocorticoids and 1,25(OH)2D3 that modulate other aspects of the osteoblast phenotype.

Animals↗

PubMed1996

Analysis of chondroprogenitor frequency and cartilage differentiation in a novel family of clonal chondrogenic rat cell lines.

We have isolated through sequential steps of subcloning a series of normal clonal cell lines enriched for chondroprogenitors that undergo differentiation in vitro from progenitors to mature chondroblasts and chondrocytes forming three-dimensional cartilage nodules. In the parental chondroblast clone RCJ 3.1C5 (C5), differentiation and cartilage formation occurred without added hormones or growth factors, but chondrogenesis could be stimulated markedly in the presence of the glucocorticoid steroid Dexamethasone (Dex). Limiting dilution analysis indicated that greater than one in ten C5 cells plated was a chondroprogenitor capable of differentiating and forming a cartilage nodule in low density cultures, but chondrogenesis was down-regulated in higher density cultures. Dex elicited a greater stimulatory effect on cartilage nodule formation when C5 cells were plated at higher rather than lower densities. Since Dex also maintained the chondrogenic potential of C5 cells passaged repeatedly, we subcloned C5 in the presence of Dex. Eight of eleven subclones were chondrogenic and the frequency of chondroprogenitors capable of cartilage formation in isolated subclones ranged from lower to much higher than in the parental C5 clone. Both Dex-independent as well as Dex-dependent clones were identified, although long-term maintenance of the chondrocyte phenotype in all subclones required Dex. These data suggest that there are Dex-dependent and Dex-independent chondroprogenitor cells, that cell-cell interactions and/or local factors can modulate cartilage nodule formation and that Dex-responsive steps are involved in long-term maintenance of chondroprogenitors in vitro. Thus, this unique family of non-transformed, clonal chondrogenic cell lines provides a quantifiable, readily manipulatable system in which cartilage differentiation and metabolism can be assessed.

Animals↗

PubMed1995

Dexamethasone alters the subpopulation make-up of rat bone marrow stromal cell cultures.

Bone marrow stromal cells comprise a heterogeneous population including fibroblastic, adipocytic, hemopoietic, and osteogenic cells. Although the conditions under which different lineages are regulated have not been fully elucidated, dexamethasone clearly stimulates osteogenic expression in stromal cultures. The purpose of this study was to begin to elucidate and quantify some of the subpopulations present when rat bone marrow stromal cells are grown with or without dexamethasone under conditions favoring bone formation. Bone marrow stromal cells from young adult rats were cultured with ascorbic acid, beta-glycerophosphate, and with or without dexamethasone for various periods of time. Culture dishes were then analyzed for cell counts, or stained with either histochemical or immunohistochemical stains, and colony types were quantitated, or cells were processed for flow cytometry. Dexamethasone significantly increased the number of alkaline phosphatase (AP) positive colonies, von Kossa positive bone nodules, alpha-naphthylbutyrate esterase positive colonies, and ED2 positive (macrophage) colonies. The number of adipocytic foci was largely unaffected in these experiments. Flow cytometry confirmed colony counts and showed stimulation by dexamethasone of AP positive cells and macrophages, and in addition, the reduction of hemopoietic cells expressing leukocyte common antigen. These data show conclusively that when rat bone marrow stromal populations are grown under conditions stimulating osteoprogenitor differentiation and bone formation, the stromal subpopulation make-up, including expression of hemopoietic lineages, is markedly altered.

Adipocytes↗

PubMed1995

Aluminum inhibits both initiation and progression of mineralization of osteoid nodules formed in differentiating rat calvaria cell cultures.

Osteoid nodules form in cultures of fetal rat calvaria (RC) cells grown in medium containing 10% fetal bovine serum (FBS) and 50 microns/ml of ascorbic acid. When 10 mM beta-glycerophosphate (beta-GP) is added, the nodules mineralize in two phases: an initiation phase that is dependent upon alkaline phosphatase activity for cleavage of beta-GP to inorganic phosphate (P(i)) and a progression phase that proceeds independently of the activity of alkaline phosphatase and does not require exogenous phosphate. We have used this system to investigate the effects of aluminum (Al3+)on mineralization. When AlCl3 was added to culture medium at concentrations of 0, 3, 10, 30, 100, and 300 muM, the total concentrations of aluminum were 0.98, 6.07, 16.82, 40.19, 88.45, and 284.52 muM, respectively. The corresponding free Al3+ concentrations, assessed after ultrafiltration, were found to be 1.11, 1.75, 3.40, 6.22, 5.38, and 12.11 muM. In cultures in which osteoid was formed and mineralization initiated in the presence of added Al+ (3-300 muM), a dose-dependent inhibition of mineralization occurred. Osteoid formed in the presence of added Al3+ mineralized normally when Al3+ was removed from cultures at the time of initiation of mineralization with beta-GP. In osteoid nodules grown in the absence of Al3+, addition of Al3+ (3-300 muM) at the start of the initiation phase of mineralization resulted in a dose-dependent inhibition of mineralization. Addition of Al3+ to cultures after mineralization had been initiated in the absence of Al3+ inhibited progression of mineralization at added Al3+ concentrations of 10 muM and above. Al3+ did not decrease the conversion of beta-GP to P(i) and caused a small but significant increase in alkaline phosphatase activity at added concentrations of 100 muM or greater. The data show that Al3+ inhibits both the initiation and progression phases of mineralization starting at added concentrations of 3-10 muM (approximately 1.7-3.4 muM free Al3+) and that mineralization of osteoid formed in the presence of Al3+ is unaffected if Al3+ is removed prior to the initiation of mineralization.

Alkaline Phosphatase↗

PubMed1995

Bone matrix proteins in osteogenesis and remodelling in the neonatal rat mandible as studied by immunolocalization of osteopontin, bone sialoprotein, alpha 2HS-glycoprotein and alkaline phosphatase.

The neonatal rat mandible was used as a model to study bone formation, mineralization, quiescence, and resorption, using immunolocalization and a variety of tissue-processing techniques. Monospecific antibodies for osteopontin (OPN), bone sialoprotein (BSP), alkaline phosphatase (AP) and alpha 2HS-glycoprotein (alpha 2HS-GP) were used on fixed paraffin-embedded tissue, fixed frozen tissue and unfixed frozen tissue. Immunostaining was correlated with mineral content by two procedures, the von Kossa and the morin techniques. Morin fluorescence was used with secondary immunostaining to provide a way of closely correlating bone matrix proteins and matrix mineralization. Co-immunolocalization procedures were used to compare the sites of bone proteins in the matrix. AP was found earliest during osteogenic cell differentiation, appearing in the preosteoblasts, followed by OPN and BSP, which first appeared in osteoblasts. alpha 2HS-GP expression was not observed in cells. The results provide clear evidence for the presence of OPN in osteoid, while BSP and alpha 2HS-GP were confined to the mineralized matrix. Immunostaining of bone proteins is highly technique-dependent: immunolocalization investigations required several methods of approach to ensure adequate demonstration of these proteins in cells and matrix. The results support the contention that osteopontin is multifunctional in bone metabolism, and that alpha 2HS-GP, though produced in the liver, is abundant in bone matrix and may also have a function in bone metabolism.

Alkaline Phosphatase↗

PubMed1995

Tri-iodothyronine (T3) and dexamethasone interact to modulate osteoprogenitor cell differentiation in fetal rat calvaria cell cultures.

We investigated the role of 3,5,3'-tri-iodothyronine (T3) in regulating differentiation of osteoprogenitor cells and also studied the effects of the glucocorticoid hormone dexamethasone (Dex) on the T3-induced effects on osteoprogenitor populations. This was done by determining the effects of either hormone alone, or of combinations of the two hormones, on the number of bone nodules formed in long-term cultures of rat calvaria cells. In this system, Dex has been shown to increase bone nodule formation, the maximal effective dose being 10 nM (Bellows et al. Endocrinology 121: 1985-1992; 1987). In standard culture medium containing 15% fetal bovine serum FBS), low concentrations of T3 (0.001-0.1 nM) had no effect on the number of bone nodules, while higher concentrations of 1-100 nM inhibited. However, in culture medium containing 10 nM Dex, the lower concentrations of T3 markedly increased the number of nodules. Short term pulse experiments with these low concentrations of T3 in the presence of Dex indicated that stimulation of nodule formation occurred only when T3 was present prior to confluency. Higher concentrations of T3 (1-100 nM) decreased nodule number whether or not Dex was added. We then cultured cells in medium containing FBS from which T3 and T4 were removed by treatment with AG-1 chi-10 resin. In both + or - Dex conditions, bone nodule formulation was increased 1.5 to 2-fold in T3, T4-depleted medium when compared with cultures maintained in standard culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗

PubMed1995

Osteoblast and chondroblast differentiation.

Recognition of discrete commitment and differentiation stages requires characterization of changes in proliferative capacity together with the temporal acquisition or loss of expression of molecular and morphological traits. Both cell lines and primary cultures have been useful for analysis of transitional steps in the chondroblast (CB) and osteoblast (OB) lineages. One striking feature is that OBs and CBs share expression of some molecules, including newer markers such as epsilon BP (galectin-3), while also having unique markers. The fact that hypertrophic chondrocytes appear able to downregulate cartilage markers and upregulate OB markers also points to an interesting lineage relationship that needs to be explored further. Recently, we have focused on the osteoprogenitors that divide and differentiate into mature OBs forming bone nodules in fetal rat calvaria cell cultures. We use cellular, immunocytochemical, and molecular approaches, including PCR on small numbers of cells, to discriminate stages. Nodule formation is characterized by loss of proliferative capacity and sequential increased marker expression, that is, alkaline phosphatase (AP), followed by bone sialoprotein (BSP), and osteocalcin. Upregulation of collagen type I and biphasic expression of osteopontin, with two peaks corresponding to proliferation and differentiation stages, also occurs. A variety of other molecules are also upregulated in the mature OB, including epsilon BP and CD44s. By replica plating and PCR, we have begun to study the expression of the messenger RNAs (mRNAs) for potential regulatory molecules (e.g., PTHrP) and their receptors (e.g., PTHR, FGFR-1, and PDGFR alpha) and have found all to be modulated during the progression from committed osteoprogenitor to mature OB.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Leukemia inhibitory factor inhibits osteogenic differentiation in rat calvaria cell cultures.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine with both anabolic and catabolic effects on bone tissue. To investigate the effect of LIF on bone formation in the absence of a resorption cycle, we used fetal rat calvaria cell cultures and quantified bone nodule production, which provides a colony assay to analyze the effects of factors on osteoprogenitor differentiation and bone formation. In these cultures, dexamethasone (Dex) stimulates bone nodule formation. In dose-response experiments, LIF inhibited bone nodule formation by cells cultured with (+Dex; ID50 = 250 U/ml) or without (-Dex; ID50 = 30 U/ml) 10(-8) M Dex. Residual nodules were small and poorly mineralized. Continuous exposure to LIF (500 U/ml) up to day 25 did not affect either the growth rate or saturation density of the cultures, but decreased alkaline phosphatase activity and bone nodule production, with greater inhibition in -Dex cultures. Exposure to LIF (500 U/ml) for 3 days early during nodule formation (about day 10) reduced bone nodule numbers to the same extent as continuous treatment in -Dex cultures and significantly, but less markedly, in +Dex cultures; earlier and later pulses had no effect. Northern blot analysis of expression of messenger RNAs of bone related proteins in cultures pulsed (-Dex) at various stages of development showed marked inhibition of alkaline phosphatase, bone sialoprotein, and osteocalcin; slight inhibition of type I collagen; early stimulation of osteopontin; and no effect on Secreted Protein, Acidic and Rich in Cysteine/osteonectin. These results suggest that LIF is an inhibitor of bone nodule formation in these cultures, acting at a stage when late osteoprogenitors and/or early osteoblasts are present, and that Dex may modulate the effects of LIF by shifting effective doses to higher concentrations.

Alkaline Phosphatase↗

PubMed1994

Carbonic anhydrase II mRNA expression in individual osteoclasts under "resorbing" and "nonresorbing" conditions.

Rabbit osteoclasts can be transformed from a nonresorbing state to a resorbing state by transferring them from culture medium at pH 7.5 to one at pH 6.5. We evaluated whether expression of mRNA for carbonic anhydrase (CA-II) could be used as an indicator of the state of activity of individual osteoclasts. A cDNA probe to rabbit carbonic anhydrase II (CA-II) was prepared and used for in situ hybridization analysis of osteoclasts isolated from neonatal rabbit long bones. Quantitation by grain counting revealed heterogeneity within the osteoclast population: osteoclasts with a "compact" (rounded, less spread) morphology expressed higher levels of CA-II mRNA than "spread" osteoclasts with similar numbers of nuclei. When maintained at pH 6.5 for 6 h, the level of CA-II mRNA was increased significantly in osteoclasts of both morphologies compared with those in parallel cultures maintained at pH 7.5. These results were confirmed by quantitating CA-II mRNA using the polymerase chain reaction (PCR). Oligonucleotide primers specific for rabbit CA-II were synthesized and used to amplify CA-II cDNA transcribed from mRNA prepared from single or small numbers (one to eight cells) of osteoclasts that were collected with a micromanipulator. This generated a approximately 510 bp PCR product, corresponding to the predicted size of the CA-II fragment encompassed by the primers. For quantitation, CA-II mRNA levels were compared with the levels of a approximately 900 bp actin fragment that was coamplified in the same reaction mixture or amplified separately in a duplicate sample of the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1994

Displacement and translocation of osteoblast-like cells by osteoclasts.

Rabbit osteoclasts and rabbit osteoblast-like stroma cells (OB cells) were placed onto plastic surfaces and the migration patterns of individual osteoclasts and osteoclast-OB interactions were analyzed with time-lapse recording. To induce directed migration, the cultures were exposed to an electrical field of 0.01 or 0.1 V/mm. At 0.1 V/mm, osteoclasts moved directly toward the anode in some cases, clearing OB cells from their path of migration. In other cases, osteoclasts migrated toward the anode for part of the time but then changed direction and moved toward groups of OB cells. Observations were made on osteoclasts interacting with single OB cells or small colonies and on osteoclasts interacting with OB monolayers, at both field strengths; the results were independent of field strength. There were several characteristic behaviors. With single OB cells and small OB colonies, retraction of OB cells upon contact with the osteoclast was the predominant mechanism whereby these cells begin to move out of the path of the osteoclast. A pronounced ruffling or blebbing of the OB cell membrane often followed retraction. When osteoclasts displaced OB cells that were part of a monolayer, extension of an osteoclast lamellipodium underneath the edge of the OB cell layer generally preceded partial retraction of the OB cells involved. It sometimes appeared as if the detached or partially detached OB cells were "pushed" by the osteoclast, which in some cases resulted in OB cells being moved hundreds of microns in a period of a few hours, at rates comparable to the normal speed for osteoclast migration (congruent to 100 microns/h), much faster than the normal speed for OB cells (congruent to 10 microns/h).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗