Search

The role of histone deacetylase 1 and 2 (HDAC1 and HDAC2) in regulating cartilage-specific gene expression was explored in primary human chondrocytes. HDAC1 and HDAC2 protein levels were elevated in chondrocytes from osteoarthritic patients, consistent with a down-regulation of some cartilage marker genes. When expressed in these cells, HDAC1 and HDAC2 repressed aggrecan and collagen 2(alpha1) expression but differed in their repression of collagen 9(alpha1), collagen 11(alpha1), dermatopontin, and cartilage oligomeric matrix protein (COMP). To identify the basis of these differences between HDAC1 and HDAC2, their carboxy-terminal domains (CTDs) were deleted, which led to proteins that retained enzymatic activity but were unable to repress cartilage gene expression. Further, exchange of the CTDs between HDAC1 and HDAC2 led to proteins that were enzymatically active but displayed altered target gene specificity, indicating that these CTDs can function independently of HDAC enzymatic activity, to target the HDACs to specific genes. The Snail transcription factor was identified as a mediator of HDAC1 and HDAC2 repression of the collagen 2(alpha1) gene, via its interaction with the HDAC1 and 2 CTDs. The data indicate that the CTD serves a novel function within HDAC1 and HDAC2, to mediate repression of cartilage-specific gene expression in human chondrocytes.

SirT1 is an NAD-dependent histone deacetylase that regulates gene expression, differentiation, development, and organism life span. Here we investigate the function of SirT1 in human chondrocytes derived from osteoarthritic patients. Elevation of SirT1 protein levels or activity in these chondrocytes led to a dramatic increase in cartilage-specific gene expression, whereas a reduction in SirT1 levels or activity significantly lowered cartilage gene expression. SirT1 associated with the cartilage-specific transcription factor Sox9, enhancing transcription from the collagen 2(alpha1) promoter in a Sox9-dependent fashion. Consistent with this association, SirT1 was targeted to the collagen 2(alpha1) enhancer and promoter, which in turn recruited the coactivators GCN5, PGC1alpha, and p300. This led to elevated marks of active chromatin within the promoter; that is, acetylated histone K9/K14 and histone H4K5 as well as trimethylated histone H3K4. Finally, alterations in the NAD salvage pathway enzyme nicotinamide phosphoribosyltransferase led to changes in NAD levels, SirT activity, and cartilage-specific gene expression in human chondrocytes. SirT1, nicotinamide phosphoribosyltransferase, and NAD may, therefore, provide a positive function in human cartilage by elevating expression of genes encoding cartilage extracellular matrix.

Several proteins with important roles in oncogenesis have been shown to regulate the function of the E2F-1 transcription factor, which is known to activate the expression of genes required for proliferation and apoptosis. Here we identify the MDMX oncoprotein as an E2F-1-binding factor, from a yeast-two hybrid screen using a portion of the E2F-1 protein as "bait." We demonstrate that the region within MDMX needed for the E2F-1:MDMX interaction is located in the central part of the protein, C-terminal of the p53-binding domain. The region within E2F-1 needed for this association is adjacent to the DNA binding domain. Further, when expressed in vivo or in vitro the MDMX protein migrates as two isoforms on SDS-PAGE, the faster migrating isoform having the stronger affinity for the E2F-1 proteins. It appears that this interaction reduces the ability of E2F-1 to bind DNA. Expression of MDMX along with E2F-1 and Dp-1 in Saos2 cells reduces the ability of E2F-1 to bind to its consensus DNA sequence, without altering E2F-1 protein levels. These data indicate that the MDMX protein is capable of associating with E2F-1 and negatively regulating its DNA binding ability., (Copyright 2002 Wiley-Liss, Inc.)

Expression of the human Na/K-ATPase beta1 subunit is regulated by a mineralocorticoid- and glucocorticoid-responsive elements. Here we identified an MR and GR responsive element, at positions -650 to -630, within the beta1 gene promoter that is required for both MR and GR activation. Independent expression of MR and GR activated by aldosterone or triamcinolone acetonide (TA) leads to significant transactivation of the beta1 promoter. Yet coexpression of both receptors activated by aldosterone plus TA or cortisol results in a much lower induction, indicating that coexpression of MR and GR is inhibitory. Gel shift mobility assay using an oligonucleotide including the 21-base pair MRE/GRE with whole cell extracts prepared from CV-1 cells overexpressing MR or GR showed specific MR and GR binding to this sequence. Additionally, antibodies to both MR and GR effectively supershifted the protein-DNA complexes, indicating that these receptors bound to the DNA sequence. Finally, the 21-base pair MRE/GRE was capable of activating transcription from a heterologous promoter in response to both aldosterone and TA. Together these data indicate that the 21-base pair sequence represents a true MRE/GRE and that optimal activation of the human Na/K-ATPase beta1 promoter is controlled by mineralocorticoid and glucocorticoid hormones. It appears that an interaction of MR with GR on the beta1 promoter effectively down-regulates transcription.

Fibronectin within the extracellular matrix plays a role in cell attachment, spreading, and shape, while it also affects aspects of cell proliferation. Transcription factors such as E2F1 are also known to regulate cell shape and cell proliferation. Yet, to date no linkage has been established between fibronectin expression and E2F1. We show here that cells constitutively expressing a mutant E2F1 protein (E2F1d87) produce reduced amounts of fibronectin mRNA and protein. The altered expression of fibronectin seen in the E2F1d87 expressing cells is due, in part, to a reduction in transcription from the fibronectin promoter. Providing exogenous fibronectin, but not Type I collagen or laminin, as a substrate for cell adhesion is sufficient to revert the altered morphology and reestablish actin-containing microfilaments lost in the mutant cell line. An additional characteristic of the cells expressing the mutant E2F1 is that they demonstrate slow growth and a doubling in S phase duration. While providing exogenous fibronectin as an adhesion substrate did not shorten the S phase duration in the mutant line, it did significantly shorten the S phase duration in the parental NIH3T3 cell line, implicating a role for the extracellular matrix in regulating S phase transit in normal cells.

Analysis of the regulatory promoter region of the human alpha 1 (I) collagen-encoding gene (COL1A1) gene indicated the presence of G+C-rich sequence elements that are potential binding sites for the transcription factor Sp1. As a step toward understanding transcriptional regulation of the human COL1A1, we examined Sp1 binding in the promoter region using DNase I footprinting, and analyzed the effect of Sp1 expression on COL1A1 promoter activity in transiently transfected Drosophila melanogaster cells in vivo. The results indicated that recombinant human Sp1 interacted specifically with two G+C-rich sequences within the COL1A1 promoter. Binding of factors in nuclear extracts prepared from human dermal fibroblasts to a 22-nucleotide deoxyribonucleotide (oligo) spanning the 5' G+C-rich sequence required Zn2+, and was abolished by excess Sp1 consensus binding site oligos, or by anti-Sp1 antibodies. Studies in which a series of progressively 5'-deleted COL1A1 promoter::cat constructs were co-expressed with an Sp1 expression plasmid in a cellular background devoid of Sp1 homology demonstrated that Sp1 markedly enhanced the COL1A1 promoter activity. These results suggest that the transcriptional activity of the human COL1A1 can be positively regulated by Sp1.

Background: Proliferation of smooth muscle cells (SMCs) plays an important role in vascular pathobiology, being involved in the development of coronary restenosis and atherosclerosis. The activation of nuclear proto-oncogenes appears to be a final common pathway onto which various mitogenic signals coverage. Accordingly, we attempted to determine whether the activation of the c-myc nuclear proto-oncogene is essential for human SMC proliferation and explored the possibility of inhibiting their growth using antisense oligonucleotides directed against c-myc messenger RNA (mRNA)., Methods and Results: Proliferation of human SMCs was associated with an increase in c-myc mRNA expression after growth stimulation. Using 15-mer phosphorothioate oligonucleotides (oligomers), we tested their growth-inhibitory effect in SMCs in vitro. Antisense oligomers directed against the translation initiation region of the human c-myc gene exhibited a significant antiproliferative effect, whereas sense and mismatched oligomers did not inhibit the growth. The growth-inhibitory effect of c-myc antisense oligomers was dose dependent and preventable by an excess of sense oligomers. Furthermore, growth inhibition of SMCs treated with c-myc antisense oligomers was associated with a marked decrease in the c-myc mRNA level. Phosphorothioate oligomers remained stable in medium containing 20% serum and were detectable in SMCs as early as 1 hour after cell exposure. Intact oligomers rapidly accumulated intracellularly and persisted within human SMCs for at least 16 hours., Conclusions: c-myc antisense oligomers reduced c-myc expression and produced a significant growth inhibition of human SMCs, indicating an important role of c-myc gene activation in the process of SMC proliferation. Furthermore, extracellular stability and rapid cellular uptake provide the basis for future studies assessing the therapeutic role of the c-myc antisense approach in reducing SMC proliferation in the process of vascular restenosis.

We have undertaken a detailed analysis of the cis-acting elements that are required for optimal transcription initiation from P2, the major promoter of the murine c-myc gene. We find that three elements contribute to promoter strength, termed ME1a2, E2F and ME1a1 at positions -85, -64 and -46 respectively (relative to P2). Individually the elements are weak, but combined they contribute to full promoter activity, all acting in a positive fashion. The E2F element is the site at which the SV40 large T antigen transactivates the c-myc promoter. However, transactivation requires the presence of the ME1a2 or ME1a1 elements in addition to E2F. By a number of criteria it appears that the ME1a2 and ME1a1 elements bind the same or a very closely related protein (monomer Mr 94,000). It was found that Hela cells contain a novel factor that is capable of binding to the ME1a1 and ME1a2 elements but only in the presence of the protein-dissociating agents deoxycholate or formamide. Finally, the E2F factor binds DNA both as a monomer and as a multiprotein complex; the latter is cell type specific. Both types of bound E2F factor form less stable protein-DNA complexes than the ME1a2/ME1a1 factor.

Interleukin-1 alpha and beta are polypeptide hormones with a broad range of biological activities. Both interleukins are recognized by a receptor that has been characterized as a member of the immunoglobin superfamily. The interleukin-1 receptor does not appear to be a tyrosine protein kinase. Moreover, the intracellular events that mediate the multiple interleukin-1 responses are poorly understood. Here we show that the JE and KC genes, first isolated and characterized as platelet-derived growth factor inducible in quiescent BALB/c-3T3 fibroblasts, are induced by femtomolar concentrations of recombinant interleukin-1 alpha (rIL-1). The response of JE and KC to IL-1 occurs at the transcriptional level. These observations suggest that an analysis of the JE and KC transcriptional response to rIL-1 may aid in identifying elements involved in interleukin-1-mediated signal transduction

Regulation of the genes by PDGF has some common features. All are primary response genes, and they can still be expressed in the presence of cycloheximide (Cochran et al. 1983; Kelly et al. 1983; Kruijer et al. 1984; Lau and Nathans 1985). In fact, many of the genes are superinduced when cells are treated with growth factors plus cycloheximide (Greenberg et al. 1986). The genes that have been characterized all contain a sequence motif in their 3'-noncoding sequences that appears to make the message labile (Meijlink et al. 1985; Treisman 1985; Shaw and Kamen 1986). All competence genes so far examined are controlled at least in part at the level of transcription (Cochran et al. 1983; Edwards et al. 1985; Almendral et al. 1988). Differences in regulation of the genes include variations in the time course of induction, ranging from 10 minutes to over 4 hours, and differences in the persistence of the mRNAs after their synthesis (Cochran et al. 1983; Muller et al. 1984; Lau and Nathans 1987). The data presented in this paper strongly suggest that multiple, distinct intracellular signals that lead to the expression of multiple genes are generated when cells are treated with growth factors such as PDGF. The variation in the time course of induction of PDGF-inducible genes suggests several models of signal transduction. Four such models are presented in Figure 5. One possibility (Fig. 5A) is that one signal is generated by the interaction of PDGF with its receptor and that this signal activates the very early genes, such as c-fos.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet-derived growth factor (PDGF) and the synthetic double-stranded RNA poly(I).poly(C) [poly(I.C)] stimulate transcription of the JE gene in BALB/c-3T3 fibroblasts. The response of JE to poly(I.C) does not appear to be channeled through any known component of the PDGF receptor signal transduction apparatus. In addition, JE sequences upstream of the transcription start site are devoid of previously identified poly(I.C)-responsive elements, such as those found in the beta-interferon gene. These data suggest that a novel signal transduction pathway regulates the JE response to PDGF and double-stranded RNA. The c-myc and c-fos proto-oncogenes also respond to this pathway but with poor efficiency. However, this pathway operates very efficiently on other PDGF-inducible genes that encode the secretory proteins KC and M-CSF.