Biomedical subjects

R Bortell

Publications and source records attributed to R Bortell.

At least 37 records · Page 2Linked to original sources

PubMed1994

Transcriptionally active nuclei isolated from intact bone reflect modified levels of gene expression in skeletal development and pathology.

Transcriptional regulation of gene expression in vivo in bone, associated with normal development or skeletal disorders, to date, has not been studied. We report the successful isolation of nuclei that are transcriptionally active from normal and osteopetrotic rat bone. Transcription rates of cell growth and bone-related genes (including histone H4, c-fos, c-jun, TGF beta 1, beta 2 macroglobulin, collagen, fibronectin, osteocalcin, osteopontin, and tartrate resistant acid phosphatase) change as a function of calvarial development from birth to 6 weeks and are selectively modified in osteopetrotic animals. Additionally, nuclei isolated from intact bone yield promoter binding factors. Bone nuclei, which transcribe faithfully and contain the normal complement of nuclear protein factors, offer a powerful approach for investigating in vivo gene regulation in skeletal development and pathology.

Animals↗

PubMed1994

Variations in vitamin D receptor transcription factor complexes associated with the osteocalcin gene vitamin D responsive element in osteoblasts and osteosarcoma cells.

Vitamin D responsive transcription of the bone-specific osteocalcin gene differs markedly in osteosarcoma cells and normal diploid osteoblasts. In osteoblasts the osteocalcin gene is transcribed, and upregulated by Vitamin D, only in post-proliferative cells, but in osteosarcoma cells expression is constitutive. This distinction in transcriptional regulation of the osteocalcin gene correlates with striking differences in the relative representation of two principal Vitamin D-dependent protein/DNA complexes designated V1 and V2 at the Vitamin D responsive element in the osteocalcin promoter. Formation of both complexes is Vitamin D dependent and they contain the Vitamin D receptor as well as an RXR related protein. Pore size exclusion and sedimentation velocity analyses suggest that the V1 and V2 complexes represent oligomeric protein assemblies (respectively, tetramers and trimers), and reflect primarily DNA-directed association of the monomeric protein components at the osteocalcin Vitamin D responsive element. UV crosslinking and methylation interference analyses of the V1 and V2 complexes at the osteocalcin Vitamin D responsive element indicate differences in protein/DNA recognition. For example, the V1 complex interacts with both steroid half-elements, whereas the V2 complex appears to recognize the proximal half-element. Our findings suggest variations in protein/protein and protein/DNA interactions of the VDR and RXR related complexes V1 and V2 at the osteocalcin Vitamin D responsive element that reflect unique properties of the osteosarcoma and normal diploid osteoblast phenotype.

Animals↗

PubMed1994

Loss of RT6 message and most circulating T cells after thymectomy of diabetes prone BB rats.

T cells expressing the RT6 surface alloantigen perform important immunoregulatory functions in the rat. Diabetes prone (DP) BB rats are deficient in circulating RT6+ T cells and develop spontaneous autoimmune diabetes mellitus. Transfusions leading to engraftment of RT6+ T cells prevent the disease. Coisogenic diabetes resistant (DR) BB rats do circulate RT6+ T cells and are free of disease. We investigated the basis for the deficiency of RT6+ T cells in the DP-BB rat and made the following observations. 1. Thymectomy causes the rapid loss of most peripheral T cells in the DP-BB rat. 2. Concomitant with the loss of T cells is the total loss of mRNA encoding RT6. 3. In contrast to the effects observed in peripheral lymphoid tissues, thymectomy does not lead to a detectable loss in RT6+ protein found in the small intestine. We conclude that the deficiency of RT6+ peripheral T cells in the DP-BB rat is due either to their short life span or to their reduced proliferative capacity following release from the thymus.

ADP Ribose Transferases↗

PubMed1993

Identification of multiple glucocorticoid receptor binding sites in the rat osteocalcin gene promoter.

The biosynthesis of osteocalcin (OC), a bone-specific, noncollagenous protein, is stringently regulated during differentiation of the osteoblast phenotype. Glucocorticoids, and also 1,25(OH)2D3, mediate the developmental regulation of OC gene transcription. In this study, we established that the -1097 to +23 promoter (pOCZCat) of the rat OC gene confers glucocorticoid responsiveness to both basal and vitamin D-induced OC expression. The presence of multiple glucocorticoid receptor (GR) binding sites in the proximal rat OC gene promoter was determined by the combined use of DNase I footprinting, dimethyl sulfate fingerprinting, and gel mobility shift analysis with glucocorticoid receptor protein. One glucocorticoid receptor binding element (GRE) resides immediately downstream of the TATA box (-16 to -1). In vivo activity was established by cotransfection of ROS 17/2.8 osteosarcoma cells with an OC-CAT construct in the presence of cloned GRE sequences (wild type or mutant) as competitors. A putative second, less protected GR binding site is located further upstream in the OC gene basal promoter within the region overlapping the TATA box. This is in direct contrast to the organization of GREs in the human OC proximal promoter wherein GR binding at the upstream GRE overlapping the TATA is stronger than at the downstream GRE. In addition, we detected sequence-specific binding of GR protein to another basal promoter element, the OC box (-99 to -76), which contains a central CCAAT motif. The presence of multiple GR binding sites in the rat OC gene proximal promoter indicates that regulation of basal and vitamin D-enhanced transcription by glucocorticoids may involve the integrated activities of multiple, independent GREs.

Animals↗

PubMed1993

Concerted control of multiple histone promoter factors during cell density inhibition of proliferation in osteosarcoma cells: reciprocal regulation of cell cycle-controlled and bone-related genes.

Cell density-induced growth inhibition of osteosarcoma cells (ROS 17/2.8) results in the shutdown of proliferation-specific histone H4 and H2B genes and the concomitant up-regulation of several osteoblast-related genes. In several respects, this reciprocal regulatory relationship is analogous to the proliferation/differentiation transition stage during development of the bone cell phenotype in normal diploid osteoblasts. Here, we comprehensively analyzed the promoter binding activities interfacing with key regulatory elements in the cell cycle-dependent histone and bone-specific osteocalcin genes. Similarly, we examined factors interacting with a series of general transcription regulatory elements that are present in a broad spectrum of promoters. The results show that histone promoter binding activities HiNF-D, HiNF-P/H4TF-2, H4UA-1, and OCT-1, as well as AP-1 activity, are proliferation dependent. These factors decline coordinately during the cessation of proliferation in both ROS 17/2.8 bone tumor cells and normal diploid osteoblasts. Collective down-regulation of these trans-activating factors occurs in both cell types within the physiological context of constitutive regulation of ubiquitous transcription factors (Sp1, ATF, and CCAAT binding proteins). In addition, during growth inhibition of ROS 17/2.8 cells we observe a complex series of modifications in protein/DNA interactions of the osteocalcin gene. These modifications include both increased and decreased representation of promoter factor complexes occurring at steroid hormone response elements as well as tissue-specific basal promoter sequences. These results demonstrate cell growth regulation of the promoter factors binding to the proliferation-specific histone and tissue-specific osteocalcin genes during the cessation of proliferation.

Animals↗

PubMed1993

Constitutive transcription of the osteocalcin gene in osteosarcoma cells is reflected by altered protein-DNA interactions at promoter regulatory elements.

The bone-specific osteocalcin (OC) gene is transcribed only after completion of proliferation in normal diploid calvarial-derived osteoblasts during extracellular matrix mineralization. In contrast, the OC gene is expressed constitutively in both proliferating and nonproliferating ROS 17/2.8 osteosarcoma cells. To address molecular mechanisms associated with these tumor-related modifications in transcriptional control, we examined sequence-specific interactions of transactivation factors at key basal and hormone-responsive elements in the OC gene promoter. In ROS 17/2.8 cells compared to normal diploid osteoblasts, the absence of a stringent requirement for cessation of proliferation to support both induction of OC transcription and steroid hormone-mediated transcriptional modulation is reflected by modifications in transcription factor binding at (i) the two primary basal regulatory elements, the OC box (which contains a CCAAT motif as a central core) and the TATA/glucocorticoid-responsive element domain, and (ii) the vitamin D-responsive element. Particularly striking are two forms of the vitamin D receptor complex that are present in proliferating osteoblasts and osteosarcoma cells. Both forms of the complex are sensitive to vitamin D receptor antibody and retinoic X receptor antibody. After the down-regulation of proliferation, only the lower molecular weight complex is found in normal diploid osteoblasts. Both forms of the complex are present in nonproliferating ROS 17/2.8 cells with increased representation of the complex exhibiting reduced electrophoretic mobility that is phosphorylation-dependent.

Animals↗

PubMed1993

Postproliferative transcription of the rat osteocalcin gene is reflected by vitamin D-responsive developmental modifications in protein-DNA interactions at basal and enhancer promoter elements.

In the osteocalcin (OC) gene promoter, both independent positive and negative regulatory elements, as well as others with contiguous [TATA/glucocorticoid-responsive elements (GRE)] or overlapping [TATA/GRE, vitamin D-responsive enhancer elements (VDRE)/AP-1, and OC box/AP-1] domains, are sites for modifications in protein-DNA interactions. In the present studies, we have examined nuclear protein extracts from fetal rat calvarial cells that undergo a developmental sequence of bone cell differentiation. Our results demonstrate modifications in protein-DNA interactions that relate to the developmental stages of the osteoblast and support developmental regulation of OC gene transcription. Basal expression of the OC gene is associated with sequence-specific protein-DNA interactions at the OC box, VDRE, and TATA/GRE box. Distinct differences are observed in proliferating osteoblasts, where the OC gene is not transcribed compared to postproliferative, differentiated osteoblasts that transcribe the OC gene. Furthermore, the protein-DNA complexes that reflect hormonal control are also developmentally regulated, mediating both the transcriptionally active and repressed states of the OC gene. For example, in proliferating osteoblasts, a vitamin D receptor-antibody-sensitive complex is formed that is different from the DNA binding complex induced by vitamin D postproliferatively when the OC gene is transcribed. Mutational analysis of the steroid hormone binding domain and the overlapping AP-1 site at the VDRE supports mutually exclusive occupancy by Fos-Jun heterodimers and vitamin D receptor. Such protein-DNA interactions at the VDRE are consistent with repression of competency for vitamin D-mediated transcriptional enhancement in proliferating osteoblasts expressing high levels of Fos and Jun.

Animals↗

PubMed1993

Inhibition of induced endochondral bone development in caffeine-treated rats.

We have addressed questions raised by the observation in fetal rats of delayed ossification induced by caffeine at maternal doses above 80 mg/kg body weight per day. The effect of caffeine on endochondral bone development and mineralization has been studied in an experimental model system of bone formation which involves implantation of demineralized bone particles (DBP) in subcutaneous pockets of young growing rats. Caffeine's effects on cellular events associated with endochondral ossification were examined directly by quantitating cellular mRNA levels of chondrocyte and osteoblast growth and differentiation markers in DBP implants from caffeine-treated rats harvested at specific stages of development (day 7 through day 15). Oral caffeine administration to rats implanted with DBP resulted in a dose dependent inhibition of the formation of cartilage tissue in the implants. Histologic examination of the implants revealed a decrease in the number of cells which were transformed to chondrocytes compared to control implants. Those cartilaginous areas that did form, however, proceeded through the normal sequelae of calcified cartilage and bone formation. At the 100 mg/kg dose, cellular levels of mRNA for histone, collagen type II, and TGF beta were all reduced by greater than 40% of control implants consistent with the histological findings. Alkaline phosphatase activity in the implants and mRNA levels for proteins reflecting the hypertrophic chondrocyte and bone phenotype, collagen type I and osteocalcin were markedly decreased compared to controls. Lower doses of 50 and 12.5 mg/kg caffeine also resulted in decreased cellular proliferation and transformation to cartilage histologically and reflected by significant inhibition of type II collagen mRNA levels (day 7). The effects of caffeine on gene expression observed in vivo during the period of bone formation (day 11 to day 15) in the DBP model were similar to the inhibited expression of H4, alkaline phosphatase, osteocalcin, and osteopontin found in fetal rat calvarial derived osteoblast cultures following 24 hour exposure of the cultures to 0.4 mM caffeine. Thus the observed delayed mineralization in the fetal skeleton associated with caffeine appears to be related to an inhibition of endochondral bone formation at the early stages of proliferation of undifferentiated mesenchymal cells to cartilage specific cells as well as at later stages of bone formation.

Administration, Oral↗

PubMed1993

Identification and characterization of two proximal elements in the rat osteocalcin gene promoter that may confer species-specific regulation.

The rat osteocalcin gene encodes a 6-kD osteoblast-specific protein that is expressed post-proliferatively. The developmental and steroid hormone responsive expression of the osteocalcin gene is transcriptionally regulated by a promoter with multiple basal and enhancer elements that exhibit activity controlled by a series of physiological mediators (e.g., 1.25(OH)2D3, glucocorticoids). In this study, we established the contribution of the rat osteocalcin (OC) box domain (-99 to -76), a proximal basal element with a CCAAT motif as a central core, to transcriptional activity of the rat osteocalcin gene with in vivo co-transfection assays. By this same assay, however, the highly homologous (22 of 24 nt) human OC box element was unable to compete for transcription factor binding with the rat OC promoter. In vitro protein/DNA interaction studies confirm the presence of two protein binding sites in the OC box region, one of which overlaps the CCAAT motif and, at least in part, accounts for species-specific expression. Competition analysis established that the single nucleotide substitution of adenine for thymine, which converts the core motif of the rat OC box (CCAAT) to the core motif of the human OC box (CCAAA), accounts for observed species differences in transcription factor interactions. The CCAAT-specific protein/DNA interactions are heat stable and insensitive to phosphatase treatment. At second protein/DNA interaction located upstream of the CCAAT motif includes two steroid-like half-elements. These interactions are heat labile and sensitive to phosphatase treatment in contrast to the CCAAT-specific interactions. The human OC promoter contains only a single steroid-like half-element, while two steroid half-elements with an 11 nucleotide spacer are present in the rat OC promoter. These observed variations in sequence organization and transactivation factor binding in analogous proximal basal regulatory regions of the OC gene promoter may provide a basis for species-restricted variations in responsiveness to physiological mediators of OC gene expression at the transcriptional level.

Animals↗

PubMed1992

Vitamin D-responsive protein-DNA interactions at multiple promoter regulatory elements that contribute to the level of rat osteocalcin gene expression.

The observation that vitamin D-mediated enhancement of osteocalcin (OC) gene expression is dependent on and reciprocally related to the level of basal gene expression suggests that an interaction of the vitamin D responsive element (VDRE) with basal regulatory elements of the OC gene promoter contributes to both basal and vitamin D-enhanced transcription. Protein-DNA interactions at the VDRE of the rat OC gene (nucleotides -466 to -437) are reflected by direct sequence-specific and antibody-sensitive binding of the endogenous vitamin D receptor present in ROS 17/2.8 osteosarcoma nuclear protein extracts. In addition, a vitamin D-responsive increase in OC gene transcription is accompanied by enhanced non-vitamin D receptor-mediated protein-DNA interactions in the "TATA" box region (nucleotides -44 to +23), which also contains a potential glucocorticoid responsive element. Evidence for proximity of the VDRE with the basal regulatory elements is provided by two features of nuclear architecture. (i) Nuclear matrix attachment elements in the rat OC gene promoter that bind nuclear matrix proteins with sequence specificity may impose structural constraints on promoter conformation. (ii) Limited micrococcal nuclease digestion and Southern blot analysis indicate that three nucleosomes can be accommodated in the sequence spanning the OC gene VDRE, the OC/CCAAT box (nucleotides -99 to -76), and the TATA/glucocorticoid responsive element, and thereby the potential distance between the VDRE and the basal regulatory elements can be reduced. A model is presented for the contribution of both the VDRE and proximal promoter elements to the enhancement of OC gene transcription in response to vitamin D. The vitamin D receptor plus accessory proteins may function cooperatively with basal regulatory factors to modulate the extent to which the OC gene is transcribed.

Animals↗

PubMed1992

Differential regulation of H4 histone gene expression in 3T3-L1 pre-adipocytes during arrest of proliferation following contact inhibition or differentiation and its modulation by TGF beta 1.

The aim of this study was to address whether there is a fundamental difference in regulation of histone gene expression in cells that have become quiescent but retain the ability to proliferate, compared with those cells that have differentiated. We compared multiple levels of regulation of histone gene expression during 3T3-L1 pre-adipocyte differentiation. Confluent cells induced to differentiate by treatment with insulin, dexamethasone, and isobutylmethylxanthine initially exhibited an increased proliferative response compared with cells given serum alone. This initial differentiation response was associated with a twofold increase in both histone gene transcription and cellular histone mRNA levels, as well as with enhanced sequence-specific binding of nuclear factors to the proximal cell-cycle-regulatory element of the H4 histone promoter. Transforming growth factor beta 1, an inhibitor of 3T3-L1 differentiation, increased both the percentage of proliferating cells and the cellular levels of histone mRNA when given in addition to serum stimulation, but no enhancement of these parameters was observed upon addition of TGF beta 1 to the differentiation treatment. Interestingly, although TGF beta 1 enhanced binding of nuclear factors to the proximal cell cycle regulatory element of the histone promoter, these protein/DNA interactions were not associated with an increase in histone transcription. Our results are consistent with the down-regulation of histone gene expression at confluency being controlled primarily at the post-transcriptional level, in contrast to an increased involvement of transcriptional down-regulation at the onset of differentiation.

3T3 Cells↗

PubMed1991

Isolation and characterization of a cDNA from a human histone H2B gene which is reciprocally expressed in relation to replication-dependent H2B histone genes during HL60 cell differentiation.

A variant human histone H2B cDNA (HHC289) has been cloned and characterized and shown to have a complex pattern of regulation with respect to the HeLa S3 cell cycle and HL60 cell differentiation. The H2B protein coding region of HHC289 is flanked at the 3' end by a 1798-nt nontranslated trailer that contains a region of hyphenated dyad symmetry and a poly(A) addition sequence, followed by a poly(A) tail. Nuclear run-on transcription analysis revealed a 2-fold increase in transcription of the HHC289 gene during S phase, in comparison to replication-dependent human histone genes which exhibit a 2-3-fold increase in transcription during S phase. Northern blot analysis indicated that the levels of the 2300-nt HHC289 mRNA species did not vary significantly during the HeLa S3 cell cycle, in comparison to replication-dependent H2B mRNAs which are elevated 15-fold during S phase. Northern blot analysis also revealed a reciprocal relationship during the onset of HL60 differentiation between the expression of the HHC289 H2B gene and the replication-dependent H2B genes. The levels of the 2300-nt HHC289 H2B species increased approximately 10-fold during HL60 cell differentiation whereas the levels of cell cycle dependent H2B mRNAs decreased to less than 1% of those in proliferating cells. These results suggest that complex transcriptional and posttranscriptional regulatory mechanisms control cellular levels of mRNAs from various human H2B histone genes during progression through the cell cycle and at the onset of differentiation.

Amino Acid Sequence↗

PubMed1991

Phenotype suppression: a postulated molecular mechanism for mediating the relationship of proliferation and differentiation by Fos/Jun interactions at AP-1 sites in steroid responsive promoter elements of tissue-specific genes.

There is a generalized reciprocal relationship between cell growth and expression of genes that occurs following completion of proliferation, which supports the progressive development of cell and tissue phenotypes. Molecular mechanisms which couple the shutdown of proliferation with initiation of tissue-specific gene transcription have been addressed experimentally in cultures of primary diploid osteoblasts that undergo a growth and differentiation developmental sequence. Evidence is presented for a model which postulates that genes transcribed post-proliferatively are suppressed during cell growth by binding of the Fos/Jun protein complex to AP-1 promoter sites associated with vitamin D responsive elements of several genes encoding osteoblast phenotype markers (Type I collagen, alkaline phosphatase, osteocalcin).

Animals↗

PubMed1991

Transcriptional element H4-site II of cell cycle regulated human H4 histone genes is a multipartite protein/DNA interaction site for factors HiNF-D, HiNF-M, and HiNF-P: involvement of phosphorylation.

Cell cycle regulated gene expression was studied by analyzing protein/DNA interactions occurring at the H4-Site II transcriptional element of H4 histone genes using several approaches. We show that this key proximal promoter element interacts with at least three distinct sequence-specific DNA binding activities, designated HiNF-D, HiNF-M, and HiNF-P. HiNF-D binds to an extended series of nucleotides, whereas HiNF-M and HiNF-P recognize sequences internal to the HiNF-D binding domain. Gel retardation assays show that HiNF-D and HiNF-M each are represented by two distinct protein/DNA complexes involving the same DNA binding activity. These results suggest that these factors are subject to post-translational modifications. Dephosphorylation experiments in vitro suggest that both electrophoretic mobility and DNA binding activities of HiNF-D and HiNF-M are sensitive to phosphatase activity. We deduce that these factors may require a basal level of phosphorylation for sequence specific binding to H4-Site II and may represent phosphoproteins occurring in putative hyper- and hypo-phosphorylated forms. Based on dramatic fluctuations in the ratio of the two distinct HiNF-D species both during hepatic development and the cell cycle in normal diploid cells, we postulate that this modification of HiNF-D is related to the cell cycle. However, in several tumor-derived and transformed cell types the putative hyperphosphorylated form of HiNF-D is constitutively present. These data suggest that deregulation of a phosphatase-sensitive post-translational modification required for HiNF-D binding is a molecular event that reflects abrogation of a mechanism controlling cell proliferation. Thus, phosphorylation and dephosphorylation of histone promoter factors may provide a basis for modulation of protein/DNA interactions and H4 histone gene transcription during the cell cycle and at the onset of quiescence and differentiation.

Base Sequence↗

PubMed1991

Developmental expression and hormonal regulation of the rat matrix Gla protein (MGP) gene in chondrogenesis and osteogenesis.

Matrix Gla protein (MGP), a vitamin K dependent protein, has recently been identified in many tissues. However, it is accumulated only in bone and cartilage suggesting that the expression of MGP may be related to the development and/or maintenance of the phenotypic properties of these tissues. We systematically evaluated MGP mRNA expression as a function of bone and cartilage development and also as regulated by vitamin D during growth and cellular differentiation. Three experimental models of cartilage and bone development were employed: an in vivo model for endochondral bone formation, as well as in primary cells of normal diploid rat chondrocyte and osteoblast cultures. MGP was expressed at the highest level during cartilage formation and calcification in vivo during endochondral bone formation. In chondrocyte cultures, MGP mRNA was present throughout the culture period but increased only after 3 weeks concomitantly with type I collagen mRNA. In osteoblast cultures, MGP mRNA was expressed during the proliferative period and exhibited increased expression during the period of matrix development. In contrast to osteocalcin (bone Gla protein), this increase was not dependent on mineralization but was related to the extent of differentiation associated with and potentially induced by extracellular matrix formation. During the proliferative period, type I collagen mRNA peaked and thereafter declined, while type I collagen protein steadily accumulated in the extracellular matrix. Constant MGP levels were maintained in the mineralization period of osteoblast differentiation in vitro which is consistent with the constant levels found during the osteogenic period of the in vivo system. MGP mRNA levels in both osteoblasts and chondrocytes in culture were significantly elevated by 1,25-(OH)2D3 (10(-8) M, 48 h) throughout the time course of cellular growth and differentiation. Interestingly, when MGP mRNA transcripts from vitamin D treated and untreated chondrocytes and osteoblasts were analyzed by high resolution Northern blot analysis, we observed two distinct species of MGP mRNA in the vitamin D treated chondrocyte cultures while all other cultures examined exhibited only a single MGP mRNA transcript. Primer extension analysis indicated a single transcription start site in both osteoblasts and chondrocytes with or without vitamin D treatment, suggesting that the lower molecular weight MGP message in vitamin D treated chondrocytes may be related to a modification in post-transcriptional processing. In conclusion, these results show that the selective accumulation of MGP in bone and cartilage tissues in vitro may be related to the development and/or maintenance of a collagenous matrix as reflected by increases in MGP mRNA during these periods.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Regulation of transcription-factor activity during growth and differentiation: involvement of the nuclear matrix in concentration and localization of promoter binding proteins.

Several lines of evidence are presented which support involvement of the nuclear matrix in regulating the transcription of two genes, histone and osteocalcin, that are reciprocally expressed during development of the osteoblast phenotype. In the 5' regulatory region of an H4 histone gene, which is expressed in proliferating osteoblasts early during the developmental/differentiation sequence, a dual role is proposed for the nuclear matrix binding domain designated NMP-1 (-589 to -730 upstream from the transcription start site). In addition to functioning as a nuclear matrix attachment site, the sequences contribute to the upregulation of histone gene transcription, potentially facilitated by concentration and localization of an 84kD ATF DNA binding protein. A homologous nuclear matrix binding domain was identified in the promoter of the osteocalcin gene, which is expressed in mature osteoblasts in an extracellular matrix undergoing mineralization. The NMP binding domain in the osteocalcin gene promoter resides contiguous to the vitamin D responsive element. Together with gene and transcription factor localization, a model is proposed whereby nuclear matrix-associated structural constraints on conformation of the osteocalcin gene promoter facilitates vitamin D responsiveness mediated by cooperativity at multiple regulatory elements.

Animals↗

PubMed1991

Disruption of the cytoskeleton with cytochalasin D induces c-fos gene expression.

The treatment of exponentially growing HeLa cells and quiescent WI-38 cells with cytochalasin D, which disrupts the cytoskeletal microfilaments, results in a rapid and marked increase in the transcription of the c-fos protooncogene with a concomitant increase in c-fos mRNA steady-state levels. Transcription of rRNA, HLA-B7, and H4 histone genes was not significantly affected by the drug treatment. These results suggest that the nucleus can respond to signals related to the structural organization of the cytoskeleton and selectively adjust the regulation of gene expression.

Cytochalasin D↗

PubMed1990

Gene expression during endochondral bone development: evidence for coordinate expression of transforming growth factor beta 1 and collagen type I.

Subcutaneous implantation of demineralized bone particles (DBP) into rats induces the formation of a bone ossicle by a tightly controlled sequence of chondro- and osteo-inductive events which are directly comparable to those which occur in normal endochondral bone development. Although the morphological and biochemical sequence associated with endochondral bone formation in this model has been well characterized, to date little information is available as to the gene regulation by which these events occur. To examine the expression of genes in this system, RNA was isolated from implants every 2 days over a time course spanning 3 to 19 days after implantation of DBP into rats. Cellular levels of mRNA transcripts of cell-growth-regulated and tissue-specific genes were examined by slot blot analysis and compared to the morphological changes occurring during formation of the ossicle. Analysis of the mRNA levels of histone H4 and c-myc, markers of proliferative activity, revealed several periods of actively proliferating cells, corresponding to 1) production of fibroprogenitor cells (day 3), 2) onset of bone formation (day 9), and 3) formation of bone marrow (day 19). The mRNA levels of collagen type II, a phenotypic marker of cartilage, peaked between days 7 and 9 post-implantation, corresponding to the appearance of chondrocytes in the implant, and rapidly declined on day 11 (to 5% of maximum value) when bone formation was observed. The peak mRNA levels of collagen type I, found in fibroblasts and osteoblasts, occurred first with the onset of bone formation (days 7-10) and again during formation of bone marrow (day 19). This study has demonstrated that the temporal patterns of mRNA expression of cartilage type II and bone type I collagens coincide with the morphological sequence in this model of endochondral bone formation. Further, the mRNA levels of transforming growth factor beta 1 (TGF beta) were compared to those of collagen types I and II; a direct temporal correlation of TGF beta mRNA levels with that of collagen type I was found throughout the developmental time course. This observation of a tightly coupled relationship between TGF beta and type I collagen mRNA levels is consistent with a functional role for TGF beta in extracellular matrix production during in vivo bone formation.

Animals↗