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PubMed1993

The interferon-stimulable response elements of two human genes detect overlapping sets of transcription factors.

We have previously reported three types of DNA-protein complexes, formed specifically with the interferon-stimulable response elements (ISRE) in the 5' flanking DNA of the interferon-inducible 6-16 and 9-27 genes, a type-I interferon-inducible early complex involving factor E (ISGF3), M and G complexes induced more slowly in response to type-I and type-II interferons, respectively and C1/C2, a constitutive complex(s). Similar complexes have been reported by others. The operationally defined band-shift complexes M, G and C1/C2 are shown here to be heterogeneous and to differ in their factor content, depending on the ISRE probe. With a 9-27 ISRE probe the M, G and C1/C2 complexes all contain the gamma subunit of ISGF3, which is present constitutively but is induced in response to IFN-alpha (to yield M) or IFN-gamma (to yield G). In contrast, a 6-16 ISRE probe forms band-shift complexes with IFN-alpha-inducible and IFN-gamma-inducible IRF1 and IRF2. With a 6-16 ISRE probe, therefore, M and G each correspond to two complexes which co-migrate in band-shift assays, one corresponding to IRF1, the other to IRF2. With this probe, the constitutive complex C1/C2 corresponds predominantly to IRF2. Consistent with this, IRF1 and IRF2 have lower affinity for the 9-27 ISRE than the 6-16 ISRE, whereas the reverse is true for E (ISGF3) and its gamma subunit. Relatively small differences in affinity appear sufficient to determine whether or not a band-shift complex is detected. In the case of IRF1 and IRF2, the different affinities for the 6-16 and 9-27 probes are dominated by a dinucleotide sequence in the centre of the 14-nucleotide 'core' ISRE. In contrast, preferential binding of E (ISGF3) by the 39-nucleotide 9-27 ISRE-containing sequence, although ISRE dependent, appears to be mediated by sequences 3' of the 'core' ISRE. Accordingly, these complexes can be simultaneously assayed using a hybrid probe consisting of the 5' flanking region and 'core' ISRE sequences from the 6-16 gene and sequences immediately 3' of the 'core' 9-27 ISRE sequence. No evidence was obtained for a modulatory role in factor binding for a pseudo-ISRE sequence close to ISRE in the 9-27 gene. The precise roles of IRF1 and IRF2 in the induction of IFN-beta and the control of interferon-inducible gene expression remain to be established.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed2001

Formation and properties of hybrid photosynthetic F1-ATPases. Demonstration of different structural requirements for stimulation and inhibition by tentoxin.

A hybrid ATPase composed of cloned chloroplast ATP synthase beta and gamma subunits (betaC and gammaC) and the cloned alpha subunit from the Rhodospirillum rubrum ATP synthase (alphaR) was assembled using solubilized inclusion bodies and a simple single-step folding procedure. The catalytic properties of the assembled alpha3Rbeta3CgammaC were compared to those of the core alpha3Cbeta3CgammaC complex of the native chloroplast coupling factor 1 (CF1) and to another recently described hybrid enzyme containing R. rubrum alpha and beta subunits and the CF1 gamma subunit (alpha3Rbeta3RgammaC). All three enzymes were similarly stimulated by dithiothreitol and inhibited by copper chloride in response to reduction and oxidation, respectively, of the disulfide bond in the chloroplast gamma subunit. In addition, all three enzymes exhibited the same concentration dependence for inhibition by the CF1 epsilon subunit. Thus the CF1 gamma subunit conferred full redox regulation and normal epsilon binding to the two hybrid enzymes. Only the native CF1 alpha3Cbeta3CgammaC complex was inhibited by tentoxin, confirming the requirement for both CF1 alpha and beta subunits for tentoxin inhibition. However, the alpha3Rbeta3CgammaC complex, like the alpha3Cbeta3CgammaC complex, was stimulated by tentoxin at concentrations in excess of 10 microm. In addition, replacement of the aspartate at position 83 in betaC with leucine resulted in the loss of stimulation in the alpha3Rbeta3CgammaC hybrid. The results indicate that both inhibition and stimulation by tentoxin require a similar structural contribution from the beta subunit, but differ in their requirements for alpha subunit structure.

Ca(2+) Mg(2+)-ATPase↗
PubMed1999

A full-length Cbfa1 gene product perturbs T-cell development and promotes lymphomagenesis in synergy with myc.

The Cbfa1/PEBP2 alpha A/AML3 gene plays an essential role in osteogenesis but is also expressed in the T-cell lineage where it has been implicated in lymphoma development as a target for retroviral insertional mutagenesis. As lymphoma cells with til-1 insertion express at least five distinct Cbfa1 isoforms, it is important to establish which, if any, have intrinsic oncogenic potential. We have generated transgenic mice in which the most abundant lymphoma isoform (G1/p57) is expressed under the control of the CD2 locus control region. Co-precipitation analysis of transgenic thymus revealed high levels of Cbfa1 protein in an abundant complex containing the binding cofactor Cbfb. CD2-Cbfa1-G1 mice displayed abnormal T-cell development, with a pronounced skew towards CD8 SP cells in the thymus and developed a low incidence of spontaneous lymphomas (6% at 12 months) with cells of similar phenotype. Strongly synergistic tumour development was seen when CD2-Cbfa1-G1 mice were crossed with lines carrying myc transgenes (CD2-myc or tamoxifen-regulatable CD2-mycER) and Cbfa1 was found to rescue expression of the CD2-myc transgene in pre-leukaemic mice. However, synergy did not appear to be due to a dominant block of myc-induced apoptosis by Cbfa1 as explanted primary tumours and cell lines from CD2-Cbfa1-G1/CD2-mycER mice showed accelerated death on induction with tamoxifen at similar rates to CD2-mycER controls. Moreover, thymocytes from preleukaemic CD2-Cbfa1-G1 mice showed reduced survival in vitro and increased sensitivity to the inhibitory effects of TGF-beta. This study demonstrates that a full-length Cbf alpha-chain gene can act as an oncogene without fusion to a heterologous protein.

Animals↗
PubMed2003

CCAAT-binding factor regulates expression of the beta1 subunit of soluble guanylyl cyclase gene in the BE2 human neuroblastoma cell line.

Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO). sGC is an obligatory heterodimer composed of alpha and beta subunits. We investigated human beta1 sGC transcriptional regulation in BE2 human neuroblastoma cells. The 5' upstream region of the beta1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs. The transcriptional start site of the beta1 sGC gene in BE2 cells was identified. The functional significance of consensus transcriptional factor binding sites proximal to the transcriptional start site was investigated by site deletions in the 800-bp promoter fragment. The elimination of CCAAT-binding factor (CBF) and growth factor independence 1 (GFI1) binding cores significantly diminished whereas deletion of the NF1 core elevated the transcription. Electrophoretic mobility-shift assay (EMSA) and Western analysis of proteins bound to biotinated EMSA probes confirmed the interaction of GFI1, CBF, and NF1 factors with the beta1 sGC promoter. Treatment of BE2 cells with genistein, known to inhibit the CBF binding to DNA, significantly reduced protein levels of beta1 sGC by inhibiting transcription. In summary, our study represents an analysis of the human beta1 sGC promoter regulation in human neuroblastoma BE2 cells and identifies CBF as a critically important factor in beta1 sGC expression.

Binding Sites↗
PubMed2004

Point mutations in the RUNX1/AML1 gene: another actor in RUNX leukemia.

The RUNX1/AML1 gene is the most frequent target for chromosomal translocation in leukemia. In addition, recent studies have demonstrated point mutations in the RUNX1 gene as another mode of genetic alteration in development of leukemia. Monoallelic germline mutations in RUNX1 result in familial platelet disorder predisposed to acute myelogenous leukemia (FPD/AML). Sporadic point mutations are frequently found in three leukemia entities: AML M0 subtype, MDS-AML, and secondary (therapy-related) MDS/AML. Therapy-related leukemias resulting from anticancer treatments are not uncommon, and the incidence of RUNX1 point mutations appears comparable to the incidence of the t(8;21) AML M2 subtype and the inv(16) AML M4Eo subtype. Half of the point mutations in M0 cases are biallelic, although the frequency varies with ethnicity. Most of the RUNX1 mutations are clustered in the Runt domain and result in defective DNA binding but active beta-subunit binding, which is consistent with three-dimensional structural findings and may explain the dominant inhibitory effects. Unlike the classical tumor suppressor genes requiring biallelic inactivation, haploinsufficient RUNX1 is apparently leukemogenic. However, RUNX1 abnormalities per se are insufficient to cause full-blown leukemia. Intensive investigation of cooperating genetic alterations should elucidate leukemic mechanisms.

Core Binding Factor Alpha 2 Subunit↗
PubMed1998

An electrophile responsive element (EpRE) regulates beta-naphthoflavone induction of the human gamma-glutamylcysteine synthetase regulatory subunit gene. Constitutive expression is mediated by an adjacent AP-1 site.

Exposure of HepG2 cells to beta-naphthoflavone (beta-NF) results in time- and dose-dependent increase in the steady-state mRNA levels for both the catalytic (GCSh) and regulatory (GCS1) subunits of gamma-glutamylcysteine synthetase (GCS) which catalyzes the rate-limiting step in the de novo synthesis of the cellular antioxidant glutathione (GSH) (Mulcahy, R. T., Wartman, M. A., Bailey, H. B., and Gipp, J. J. (1997) J. Biol. Chem. 272, 7445-7454). Cloning and sequencing of the GCS1 promoter region is reported. Regulatory sequences mediating basal and beta-NF induced expression of the GCSl gene were identified using a series of promoter/reporter fusion genes transfected into HepG2 cells. Sequences directing basal and beta-NF induced expression were localized between nucleotides -344 and -242 (numbered relative to the translation start site). Mutational analyses indicate that basal expression of the GCSl gene is directed by a consensus AP-1-binding site located 33 base pairs upstream of a consensus electrophile responsive element (EpRE) sequence; both cis-elements are capable of supporting beta-NF inducibility. Elimination of the inducible response requires simultaneous mutation of both sequences, however, in the presence of an intact EpRE the upstream AP-1 site is irrelevant to induction. Regulation of expression of both human GCS subunit genes in response to beta-NF is therefore mediated by cis-elements satisfying the consensus core EpRE motif.

Antioxidants↗
PubMed1997

The two alpha subunits of Escherichia coli RNA polymerase are asymmetrically arranged and contact different halves of the DNA upstream element.

RNA polymerase core enzyme of Escherichia coli is composed of two alpha subunits and one each of the beta and beta' subunits. The C-terminal domain of the RNA polymerase alpha subunit plays a key role in molecular communications with class I transcription factors and upstream (UP) elements of promoter DNA, using the same protein surface. To identify possible differences in the functional roles of the two alpha subunits, we have developed a reconstitution method for hybrid RNA polymerases containing two distinct alpha subunit derivatives in a defined orientation ("oriented alpha-heterodimer"). The binding sites of two alpha C-terminal domains on the UP element DNA were determined by hydroxyl radical-based DNA cleavage mediated by (p-bromoacetamidobenzyl)-EDTA x Fe, which was bound at Cys-269 on the UP recognition surface of one or both alpha subunits. The results clearly indicated that the two alpha subunits bind in tandem to two helix turns of the rrnBP1 UP element, and that the beta'-associated alpha subunit is bound to the promoter-distal region.

Binding Sites↗
PubMed1996

Identification of an element crucial for the sub-synaptic expression of the acetylcholine receptor epsilon-subunit gene.

The adult neuromuscular junction displays an accumulation of both the acetylcholine receptor (AChR) protein in the subneural domain of the post-synaptic membrane and the mRNAs coding for all its subunits at the level of the subjunctional "fundamental nuclei." In the course of end plate development, the epsilon-subunit, at variance with other subunits, becomes exclusively expressed at the level of the fundamental nuclei, yet at a rather late stage (around birth). To analyze the promoter region of the epsilon-subunit gene which directs its specific expression at the synapse, we used a quantitative transient in vivo expression assay in intact muscle tissue using constructs of the epsilon-subunit promoter placed upstream of the beta-galactosidase reporter gene. One crucial element for synapse-specific expression was detected between the -11 and -6 positions. Disruption of this element, either by a scanning mutation or single base mutations, greatly diminishes, or even completely inhibits, preferential expression of the transgene at the end plate. Gel shift experiments reveal the presence of a complex in nuclear muscle extracts that bind the core sequence of this element. The identification of such a site opens the possibility to identify regulatory factors responsible for compartmentalized expression at the neuromuscular junction.

Aging↗
PubMed2005

BMP-2 and insulin-like growth factor-I mediate Osterix (Osx) expression in human mesenchymal stem cells via the MAPK and protein kinase D signaling pathways.

Genetic studies place the transcription factor Osterix (Osx) downstream of Runx2, but limited information is available about Osx regulation during osteoblastic differentiation. An important role for bone morphogenetic protein-2 (BMP-2) and insulin-like growth factor-I (IGF-I) on Osx expression and the requirement for p38 for the BMP-2-mediated effect was reported previously by our group. In this study, we continued to investigate the molecular mechanisms by which BMP-2 and IGF-1 regulate Osx expression during osteoblast lineage progression. IGF-I-mediated Osx expression required all three MAPK components (Erk, p38, and JNK), whereas BMP-2 required p38 and JNK signaling. As a common mediator of growth factor signaling, we also investigated the involvement of protein kinase C/D (PKC/D) signaling. BMP-2- and IGF-I-mediated Osx expression was blocked in response to a PKD inhibitor. A selective inhibitor of conventional PKCs had no effect on the BMP-2-mediated Osx expression. BMP-2 and IGF-I induced a selective phosphorylation of PKD, and PKD was required for mineralization. PKC/D and MAPK signaling also mediate Runx2 activity. Therefore, to document the implication for Runx2 in Osx regulation, we blocked Runx2 activity using a dominant negative Runx2 construct and an ubiquitination mediator for Runx2 degradation. We showed that blocking Runx2 activity inhibited the BMP-2-mediated induction of Osx. These studies implicated that multiple signaling pathways mediate Osx, a critical gene for osteoblast differentiation and bone formation. In addition to Runx2, other signaling components may be necessary to regulate Osx during osteoblast lineage progression.

Animals↗
PubMed2005

Runx1/AML1/Cbfa2 mediates onset of mesenchymal cell differentiation toward chondrogenesis.

UNLABELLED: Runx proteins mediate skeletal development. We studied the regulation of Runx1 during chondrocyte differentiation by real-time RT-PCR and its function during chondrogenesis using overexpression and RNA interference. Runx1 induces mesenchymal stem cell commitment to the early stages of chondrogenesis. INTRODUCTION: Runx1 and Runx2 are co-expressed in limb bud cell condensations that undergo both cartilage and bone differentiation during murine development. However, the cooperative and/or compensatory effects these factors exert on skeletal formation have yet to be elucidated. MATERIALS AND METHODS: Runx1/Cbfa2 and Runx2/Cbfa1 were examined at different stages of embryonic development by immunohistochemistry. In vitro studies used mouse embryonic limb bud cells and assessed Runx expressions by immunohistochemistry and real-time RT-PCR in the presence and absence of TGFbeta and BMP2. Runx1 was overexpressed in mesenchymal cell progenitors using retroviral infection. RESULTS: Immunohistochemistry showed that Runx1 and Runx2 are co-expressed in undifferentiated mesenchyme, had similar levels in chondrocytes undergoing transition from proliferation to hypertrophy, and that there was primarily Runx2 expression in hypertrophic chondrocytes. Overall, the expression of Runx1 remained significantly higher than Runx2 mRNA levels during early limb bud cell maturation. Treatment of limb bud micromass cultures with BMP2 resulted in early induction of both Runx1 and Runx2. However, upregulation of Runx2 by BMP2 was sustained, whereas Runx1 decreased in later time-points when type X collagen was induced. Although TGFbeta potently inhibits Runx2 and type X collagen, it induces type II collagen mRNA and mildly but significantly inhibits Runx1 isoforms in the early stages of chondrogenesis. Virus-mediated overexpression of Runx1 in mouse embryonic mesenchymal cells resulted in a potent induction of the early chondrocyte differentiation markers but not the hypertrophy marker, type X collagen. Knockdown or Runx1 potently inhibits type II collagen, alkaline phosphatase, and Runx2 and has a late inhibitory effect on type X collagen. CONCLUSION: These findings show a distinct and sustained role for Runx proteins in chondrogenesis and subsequent chondrocyte maturation. Runx1 is highly expressed during chondrogenesis in comparison with Runx2, and Runx1 gain of functions stimulated this process. Thus, the Runx genes are uniquely expressed and have distinct roles during skeletal development.

Alcian Blue↗
PubMed1998

Molecular characterization and chromosomal localization of a third alpha-class hypoxia inducible factor subunit, HIF3alpha.

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors that regulate a number of adaptive responses to low oxygen tension. They are composed of alpha- and beta-subunits that belong to the basic helix-loop-helix-PAS (bHLH-PAS) superfamily. In our efforts to identify new bHLH-PAS proteins, we cloned a cDNA encoding a novel alpha-class hypoxia inducible factor, HIF3alpha. The HIF3alpha open reading frame encodes a 662-amino acid protein with a predicted molecular weight of 73 kDa and is expressed in adult thymus, lung, brain, heart, and kidney. The N-terminal bHLH-PAS domain of this protein shares amino acid sequence identity with that of HIF1alpha and HIF2alpha (57% and 53% identity, respectively). The C-terminus of HIF3alpha contains a 36-amino acid sequence that shares 61% identity with the hypoxia responsive domain-1 (HRD1) of HIF1alpha. In transient transfections, this domain confers hypoxia responsiveness when linked to a heterologous transactivation domain. In vitro studies reveal that HIF3alpha dimerizes with a prototype beta-class subunit, ARNT, and that the resultant heterodimer recognizes the hypoxia responsive element (HRE) core sequence, TACGTG. Transient transfection experiments demonstrate that the HIF3alpha-ARNT interaction can occur in vivo, and that the activity of HIF3alpha is upregulated in response to cobalt chloride or low oxygen tension.

Amino Acid Sequence↗
PubMed1996

Binding of the sigma 70 protein to the core subunits of Escherichia coli RNA polymerase, studied by iron-EDTA protein footprinting.

We have used a nonspecific protein cleaving reagent to map the interactions between subunits of the multisubunit enzyme RNA polymerase (Escherichia coli). We developed suitable conditions for using an untethered Fe-EDTA reagent, which does not bind significantly to proteins. Comparison of the cleaved fragments of the subunits from the core enzyme (alpha 2 beta beta') and the holoenzyme (core+sigma 70) shows that absence of the sigma 70 subunit is associated with the appearance of several cleavage sites on the subunits beta (within 10 residues of sequence positions 745, 764, 795, and 812) and beta' (within 10 residues of sequence positions 581, 613, and 728). A cleavage site near beta residue 604 is present in the holoenzyme but absent in the core, demonstrating that a conformational change occurs when sigma 70 binds. No differences are observed for the alpha subunit.

Amino Acid Sequence↗
PubMed2003

Enhanced expression of Runx2/PEBP2alphaA/CBFA1/AML3 during fracture healing.

The cause of the dramatic increase in expression of the osteopontin gene during fracture healing was studied in a mouse experimental model. Semiquantitative reverse transcription-polymerase chain reaction, Northern blotting, and in situ hybridization analysis showed that the enhanced expression took place prior to callus formation. The change in the expression pattern of collagenous and noncollagenous bone matrix proteins in addition to Ets-1 and Runx2, major transcription factors of osteopontin, were examined and compared to that of osteopontin. Although Ets-1 expression showed no significant change during fracture healing, enhanced expression of Runx2 corresponding to that of osteopontin was observed. Furthermore, in situ hybridization demonstrated that osteopontin-expressing cells also express the Runx2 gene. The results indicated the possibility that Runx2 is a major regulator of osteopontin during fracture healing.

Animals↗
PubMed2004

Homeobox protein MSX2 acts as a molecular defense mechanism for preventing ossification in ligament fibroblasts.

Ligaments and tendons are comprised of tough yet flexible connective tissue. Little is known, however, about the precise characteristics of the cells in ligaments and tendons due to the absence of specific markers and cell lines. We recently reported a periodontal ligament cell line, PDL-L2, with suppressed Runx2/Osf2 transcriptional activity and an inability to form mineralized nodules. The present study demonstrates that the homeobox protein Msx2 is a key factor in suppressing those two functions. Msx2 colocalizes with Runx2/Osf2 and suppresses its activity cooperatively, acting with another corepressor, TLE1, as a complex to recruit histone deacetylase 1 activity. Reverse transcription-PCR and in situ hybridization demonstrated that Msx2 expression is higher in periodontal ligament and tendon cells than in osteoblasts. Stable reduction of Msx2 expression in PDL-L2 cells induces osteoblastic differentiation, thereby causing matrix mineralization. Conversely, stable, forced Msx2 expression in MC3T3-E1 cells prevented osteoblast differentiation and matrix mineralization. Msx2-induced suppression of osteoblast differentiation was repressed by bone morphogenetic protein 2. In addition, Msx2 was downregulated in a symptom- and calcification-dependent manner at the affected region in patients with ossification of the posterior longitudinal ligament. Our findings indicate that Msx2 plays a central role in preventing ligaments and tendons from mineralizing.

Aged↗
PubMed2001

Molecular insights into the lineage-specific determination of odontoblasts: the role of Cbfa1.

The role of stable transcription complexes in initiating and consolidating programs of gene expression during lineage specification has been extensively studied. Despite the progress made in the identification of key molecules of tooth initiation and patterning, the mechanisms leading to cell differentiation during odontogenesis are unknown. Odontoblasts are exclusive dentin-producing cells that are phenotypically and functionally distinct from osteoblasts. However, not much is known about the precise determinants of odontoblast terminal differentiation--in particular, how the fate of these cells becomes delineated from that of osteogenic mesenchyme. Cbfa1(-/-) mice completely lack osteoblasts and bone, while tooth development arrests at the time of odontoblast differentiation. The purpose of this paper is to overview our studies on the role of Cbfa1 in odontoblast determination and differentiation using the Cbfa1(-/-) mouse model and various experimental approaches. Our expression analyses confirm the down-regulation of Cbfa1 expression in newly differentiated and functional odontoblasts. Second, we demonstrate that Cbfa1(-/-) incisor organs arrest at a later stage than molars, and that alpha 1 (I) collagen, a marker of odontoblast differentiation shared in common with osteoblasts, is not significantly affected by the absence of the transcription factor. Interestingly, Dspp expression in Cbfa1(-/-) appeared markedly down-regulated in putative odontoblasts. The overexpression of Cbfa1 in an odontoblast cell line (MDPC-23) results in the selective down-regulation of Dspp and not type I collagen. It is likely that, in addition to its influence on tooth epithelial morphogenesis, Cbfa1 plays a non-redundant and stage-specific role in the lineage determination and terminal differentiation of odontoblasts from dental papilla mesenchyme.

Animals↗
PubMedDate not supplied

Initiation and regulation mechanisms of ribosomal RNA transcription in the eukaryote Acanthamoeba castellanii.

Acanthamoeba rRNA transcription involves the binding of a transcription initiation factor (TIF) to the core promoter of rDNA to form the preinitiation complex. This complex is formed in the absence of RNA polymerase I, and persists for multiple rounds of initiation. Polymerase I next binds to form the initiation complex. This binding is DNA sequence-independent, and is directed by protein-protein contacts with TIF. DNA melting occurs in a separate step. In contrast to most prokaryotic transcription, melting occurs only following nucleotide addition and beta-gamma hydrolysis of ATP is not required as for polymerase II. Growth-dependent regulation of rRNA transcription is accomplished by modification of RNA polymerase I. The inactive form of polymerase (PolE) is unable to bind to the promoter and has altered heat stability. PolE is still active in elongation; thus, the modification affects the polymerase site involved in TIF contact. Modification of a polymerases I and III common subunit has been detected leading to the suggestion that transcription of stable RNAs of the ribosome might be co-regulated by this mechanism.

Acanthamoeba↗
PubMedDate not supplied

In vitro differentiation and mineralization of human dental pulp cells induced by dentin extract.

In this study, the progenitor cells isolated from the human dental pulp were used to study the effects of ethylenediaminetetraacetic acid-soluble dentin extract (DE) on their differentiation and mineralization to better understand tissue injury and repair in the tooth. Mineralization of the matrix was increasingly evident at 14, 21, and 28 d after treatment with a mineralization supplement (MS) (ascorbic acid [AA], beta-glycerophosphate [beta-GP]) and MS + DE. Real-time polymerase chain reaction results showed type I collagen upregulation after the addition of MS + DE at 7 d. Alkaline phosphatase was downregulated after the mineralization became obvious at 14 d. Bone sialoprotein was shown to be upregulated in the mineralized cell groups at all time points and dentin sialophosphoprotein after 7 d. Core binding factor a 1 was upregulated by the treatment of MS and DE at 7, 14, and 21 d. These results indicated that the MS of AA, beta-GP, and DE synergistically induced cell differentiation of pulp progenitor cells into odontoblast-like cells and induced in vitro mineralization.

Alkaline Phosphatase↗
PubMed2000

Adenoviral expression of p53 represses telomerase activity through down-regulation of human telomerase reverse transcriptase transcription.

Telomerase activation is a critical step in cellular immortality and oncogenesis. The activity of telomerase is known to be correlated with cell proliferation, but its regulation by cell cycle regulators is not well understood. In the present study, we examined the effects of p53 on telomerase activity. Wild-type p53 was introduced into SiHa cells via a recombinant adenoviral vector, Ad5CMV-p53, and change in telomerase activity was examined by quantitative telomerase assay. Telomerase activity in the Ad5CMV-p53-infected cells was significantly repressed 36 h after infection following down-regulation of human telomerase catalytic subunit [human telomerase reverse transcriptase (hTERT)] mRNA expression, whereas no change in telomerase activity was observed in the cells infected with control vector AdSCMV-beta-gal. Interestingly, repression of telomerase activity was an early event that preceded cell growth inhibition or apoptosis induced by p53 overexpression, suggesting that p53 directly regulates telomerase activity. Transient expression assays using hTERT-promoter reporter constructs revealed that overexpression of p53 significantly repressed promoter activity of hTERT. 5'-Truncation of the promoter sequences revealed that the proximal core promoter region containing multiple binding sites for transcription factor Spl was responsible for p53-mediated transcriptional repression. Mutations in these binding sites for Spl led to failure of p53 to repress transcription. These findings suggest that p53 repressed telomerase activity through down-regulation of hTERT transcription and that interaction of p53 with Sp1 or other transcription factors may be involved in this regulation.

Adenoviridae↗