Heterogeneity of cell death.

Cell death constitutes a number of heterogeneous processes. Despite the dynamic nature of cell death, studies of cell death have primarily focused on apoptosis, and cell death has often been viewed as static events occurring in linear pathways. In this article we review cell death heterogeneity with specific focus on 4 aspects of cell death: the type of cell death; how it is induced; its mechanism(s); the results of cell death, and the implications of cell death heterogeneity for both basic and clinical research. This specifically reveals that cell death occurs in multiple overlapping forms that simultaneously occur within a population. Network and pathway heterogeneity in cell death is also discussed. Failure to integrate cell death heterogeneity within analyses can lead to inaccurate predictions of the amount of cell death that takes place in a tumor. Similarly, many molecular methods employed in cell death studies homogenize a population removing heterogeneity between individual cells and can be deceiving. Finally, and most importantly, cell death heterogeneity is linked to the formation of new genome systems through induction of aneuploidy and genome chaos (rapid genome reorganization).

Silent brain infarcts, leukoaraiosis, and long-term prognosis in young ischemic stroke patients.

OBJECTIVE: To investigate prognostic relevance of silent brain infarcts (SBIs) and leukoaraiosis (LA) in young patients with ischemic stroke. METHODS: This observational cohort study included consecutive MRI-scanned patients aged 15 to 49 with first-ever ischemic stroke treated at Helsinki University Central Hospital (1994-2007) with long-term follow-up data available. Outcome measures were 1) nonfatal or fatal ischemic stroke, 2) composite vascular endpoint, and 3) death from any cause. Trial of ORG 10172 in Acute Stroke Treatment (TOAST) and Bamford criteria allowed for stroke subtyping. Number of SBIs was categorized into none, single, or multiple. LA fell into groups of none, mild, or moderate to severe (validated visual rating scale). RESULTS: The 655 patients (mean age 40.0 ± 8.0 years) included were followed for a mean 8.7 ± 3.8 years (survivors). Of the 86 (13.1%) patients with SBIs, 46 had single and 40 had multiple SBIs. In the 50 (7.6%) patients with LA, these changes were mild in 21 and moderate to severe in 29. In Cox regression analysis, multiple SBIs independently raised the risk for recurrent ischemic stroke (odds ratio 2.48; 95% confidence interval 1.24-4.94) adjusted for age, gender, risk factors, stroke etiology, and LA. After further adjustment for initial stroke severity, TOAST and Bamford subgroups, and presence of SBIs, moderate to severe LA increased the risk for death (3.43; 1.58-7.42). Neither SBIs nor LA associated with the composite vascular endpoint. CONCLUSIONS: MRI-defined SBIs and LA are prognostically valuable in young adults after their first-ever ischemic stroke.

Silent brain infarcts and leukoaraiosis in young adults with first-ever ischemic stroke.

BACKGROUND: We recently observed that 13% of 1,008 consecutive adults aged 15-49 years with first-ever ischemic stroke had one or more silent brain infarcts (SBIs), and more than 5% presented with leukoaraiosis on CT or MRI. We sought to investigate the features of and risk factors for magnetic resonance (MR)-defined SBIs and leukoaraiosis in these patients. METHODS: We analyzed the radiologic features of SBIs and leukoaraiosis in MR-scanned patients (n = 669) blinded to clinical data and examined their relation with subtype of the overt stroke. We used logistic regression to identify factors predisposing to SBIs and leukoaraiosis. RESULTS: Of the 669 patients included, 86 (13%) had SBIs, 50 (7%) had leukoaraiosis, 17 (3%) had both, and 550 had no SBIs or leukoaraiosis and served as controls. The majority (54%) had a single SBI, 20% had two SBIs, and 27% had three or more SBIs. Most SBIs were located in basal ganglia (39%) or subcortical regions (21%), but cerebellar SBIs also were rather frequent (15%). Leukoaraiosis was mainly mild to moderate. Independent risk factors for SBIs were type 1 diabetes (odds ratio [OR] 5.78, 95% confidence interval 2.37-14.10), obesity (OR 2.12, 1.07-4.19), smoking (OR 1.69, 1.05-2.72), and increasing age (OR 1.08, 1.04-1.13). Risk factors for leukoaraiosis were type 1 diabetes (OR 9.75, 3.39-28.04), obesity (OR 2.42, 1.04-5.68), female sex (OR 2.25, 1.16-4.34), and increasing age (OR 1.19, 1.10-1.29). Small-vessel disease was the predominant cause of stroke in both those with SBIs (31%) and leukoaraiosis (44%). CONCLUSIONS: Silent brain infarcts and leukoaraiosis are not uncommon among young stroke patients--type 1 diabetes being the strongest risk factor.

cDNA cloning and embryonic expression of mouse nuclear pore membrane glycoprotein 210 mRNA.

BACKGROUND: In embryonic kidneys, mesenchymal cells convert into epithelium in response to an induction by the tip of the ureter bud. Metanephric mesenchyme can also be induced to convert into epithelium in vitro. It is a model system to identify genes that could be important for epithelial development. METHODS: By differential screening of a cDNA library made from mesenchymes induced in transfilter cultures by embryonic spinal cord for 24 hours, we selected cDNA clones representing genes that were preferentially expressed in 24-hour-induced mesenchyme and not in uninduced mesenchyme. The sequence of one clone was determined and used to obtain the sequence of a complete open reading frame. By Northern blotting and in situ hybridization, the expression of the mRNA in embryonic kidneys was determined. RESULTS: We report the sequence and expression pattern of a marker for the 24-hour-induced state, mouse nuclear pore membrane glycoprotein 210 (mPOM210). The deduced 1886 amino acid sequence shows a 95% identity to the sequence of rat gp210. Northern blotting revealed a single 7.5 kb mRNA in 24-hour-induced mesenchyme, whereas message levels were fourfold to fivefold lower in uninduced mesenchyme. In situ hybridization of in vivo development confirmed the preferential expression of mPOM210 in epithelial cells. In the kidney, expression was seen in both the epithelium derived from the ureteric tree and the mesenchyme-derived epithelium. In other tissues of 13-day-old embryos, expression was also confined to the epithelium. In nervous tissues, the olfactory epithelium and walls of the lateral ventricle were the most prominently stained. Weak expression was seen in the heart. CONCLUSIONS: mPOM210 mRNA is an early marker for developing epithelial cells. Furthermore, our results suggest that nuclear pore membrane proteins could be more cell-type specific than previously anticipated.

p300/cAMP-responsive element-binding protein interactions with ets-1 and ets-2 in the transcriptional activation of the human stromelysin promoter.

In this paper we show that transcription factors Ets-1 and Ets-2 recruit transcription adapter proteins p300 and CBP (cAMP-responsive element-binding protein) during the transcriptional activation of the human stromelysin promoter, which contains palindromic Ets-binding sites. Ets-2 and p300/CBP exist as a complex in vivo. Two regions of p300/CBP between amino acids (a.a.) 328 and 596 and a. a. 1678 and 2370 independently can interact with Ets-1 and Ets-2 in vitro and in vivo. Both these regions of p300/CBP bind to the transactivation domain of Ets-2, whereas the C-terminal region binds only to the DNA binding domain of Ets-2. The N- and the C-terminal regions of CBP (a.a. 1-1097 and 1678-2442, respectively) which lack histone acetylation activity independently are capable of coactivating Ets-2. Other Ets family transcription factors failed to cooperate with p300/CBP in stimulating the stromelysin promoter. The LXXLL sequence, reported to be important in receptor-coactivator interactions, does not appear to play a role in the interaction of Ets-2 with p300/CBP. Previous studies have shown that the stimulation of transcriptional activation activity of Ets-2 requires phosphorylation of threonine 72 by the Ras/mitogen-activated protein kinase signaling pathway. We show that mutation of this site does not affect its capacity to bind to and to cooperate with p300/CBP.

Cloning and characterization of a novel matrix metalloproteinase (MMP), CMMP, from chicken embryo fibroblasts. CMMP, Xenopus XMMP, and human MMP19 have a conserved unique cysteine in the catalytic domain.

We cloned a novel matrix metalloproteinase (MMP) called CMMP from cultured primary chicken embryo fibroblasts. The cDNA-derived CMMP sequence contains 472 amino acids including a putative 19-residue signal peptide and a unique cysteine in the catalytic domain, an insertion in a sequence motif that binds the structural (noncatalytic) zinc of MMPs. Strikingly, a homologously inserted cysteine is also found in Xenopus XMMP and human MMP19, two recently cloned novel members of the MMP family. Phylogenetic analysis suggest that XMMP and MMP19 represent founding members of the MMP family, whereas CMMP is related to collagenase MMPs. Bacterially produced recombinant CMMP (without the amino-terminal inhibition domain), which was autoproteolyzed at the carboxyl-terminal domain, digested casein and gelatin. As shown by Northern blotting, CMMP mRNA of 1.8 kilobase pairs was constitutively expressed in cultured primary chicken embryo fibroblasts and up-regulated by tumor necrosis factor-alpha and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, but it was not regulated by interleukin-1, basic fibroblast growth factor, or retinoic acid. CMMP mRNA of 1.8 kb was also detected in the head and body of 8-day-old chicken embryos and dramatically up-regulated in 9-day-old embryos.

Cloning and developmental regulation of tissue inhibitor of metalloproteinases-3 (TIMP3) in Xenopus laevis early embryos.

We cloned a cDNA encoding tissue inhibitor of metalloproteinases-3 (TIMP3) from the frog Xenopus laevis. Similar to TIMP3 from other species, Xenopus TIMP3 has 188 residues including 12 conserved cysteines and Asn184, a putative site for N-linked sugars. Xenopus TIMP3 is 84% identical with human TIMP3. As shown by Northern blotting and RT-PCR, Xenopus TIMP3 mRNA is maternally inherited in eggs and midblastula (stage 8) embryos, downregulated in gastrula and then upregulated in neurula and pretailbud embryos. In select adult tissues, TIMP3 mRNA is present in heart, muscle, liver, skin, intestine and ovaries. These results suggest that TIMP3 is involved in the regulation of expression of matrix metalloproteinases in Xenopus early development and adult tissue remodeling.

A novel matrix metalloproteinase gene (XMMP) encoding vitronectin-like motifs is transiently expressed in Xenopus laevis early embryo development.

To study the role of matrix metalloproteinases (MMPs) in early vertebrate development, we cloned cDNAs for six different MMPs from the frog Xenopus laevis embryos at different stages of development and describe here a novel MMP called XMMP. Xenopus XMMP has 604 amino acids including a putative signal peptide of 22 residues. At the carboxyl-terminal end of the propeptide, XMMP has a 37-amino acid-long insertion domain containing a segment that is 38% identical with a rat vitronectin sequence between residues 108-135. Following this domain is an RRKR motif, a putative cleavage site for intracellular activation by furin proteinases. XMMP lacks a proline-rich linker peptide, or hinge region, typically found in other MMPs between the catalytic domain and carboxyl-terminal "hemopexin/vitronectin-like" domain. In XMMP, the carboxyl-terminal domain is composed of four tandem repeats that are 21-33% identical to a sequence (residues 213-264) encoded by vitronectin exon-5. Interestingly, XMMP gene is transiently expressed during Xenopus embryo development. XMMP mRNA of 3.0 kilobase pairs was undetected in the blastula stage embryo, induced in gastrula embryo, expressed in neurula embryo, and then down-regulated in pretailbud embryo. In comparison, other Xenopus MMP genes that we have cloned show a different developmental regulation. In blastula embryo, the only MMP gene expressed was found to be 92-kDa type IV collagenase, which was also expressed in the gastrula, neurula, and pretailbud embryos. Expression of stromelysin-1, stromelysin-3, and two different membrane type-MMPs was first detected in the neurula and pretailbud embryos. These results suggest that MMPs and the novel XMMP reported here play a role in Xenopus early development.

Erg, an Ets-family member, differentially regulates human collagenase1 (MMP1) and stromelysin1 (MMP3) gene expression by physically interacting with the Fos/Jun complex.

Collagenase1 (MMP1) and stromelysin1 (MMP3) are extracellular proteolytic enzymes that degrade connective tissue macromolecules and basement membranes. Both genes are regulated by the Ets and Fos/Jun families of transcription factors/oncoproteins. Here, we show that two members of the Ets-family, Ets2 and Erg and their combinations differentially regulate collagenase1 and stromelysin1 promoter activity. In transiently transfected cells, Ets2 activates both promoters whereas Erg induces collagenase1 but not stromelysin1 promoter activity. Moreover, Erg completely inhibits stromelysin1 promoter activation by Ets2. In gel shift assays however, the Erg protein bound little or not to the collagenase1 promoter, whereas it bound to the stromelysin1 promoter. By site-specific mutagenesis, we identified one major site at -88 that abolished collagenase1 promoter activation by Erg. Surprisingly, mutation of the collagenase1 AP1 site at -73 also abolished the activation by Erg suggesting that Erg cooperates with Fos/Jun in collagenase1 promoter regulation. Indeed, gel shift and in vitro protein interaction studies showed that Erg binds to the Fos/Jun complex. Thus, Erg represents the first example of a transcription factor that can distinguish between the collagenase1 and stromelysin1 promoters in that when Erg is recruited by Fos/Jun at the promoter, it transcriptionally activates collagenase1 gene but not stromelysin1 expression.

Carbohydrate-mediated regulation of matrix metalloproteinase-2 activation in normal human fibroblasts and fibrosarcoma cells.

Matrix metalloproteinase-2 (MMP-2) is activated on the cell surface by membrane type 1-MMP (MT1-MMP). Activation of proMMP-2 is induced in vitro by concanavalin A (ConA). The regulation of proMMP-2 activation is, however, not yet fully understood. We investigated the effect of plant lectins, carbohydrates and inhibitors of the cytoskeleton on proMMP-2 activation in normal (HLF1) and malignant fibroblast (HT1080) cells. Native ConA induced proMMP-2 activation in both cell types while dimeric succinyl-ConA had no effect, suggesting that receptor clustering is involved in activation. Wheat germ agglutinin (WGA) also induced proMMP-2 activation. N-acetyl-D-glucosamine (GlcNac) inhibited the effects of ConA and WGA while mannose only inhibited ConA-induced proMMP-2 activation. Mannose also inhibited the expression of MT1-MMP mRNA induced by ConA. Cytochalasin B and colchicine had no effect on the ConA induction of proMMP-2 activation. These studies help to define some of the cellular and molecular mechanisms for the induction of proMMP-2 activation.

Cloning and developmental expression of a membrane-type matrix metalloproteinase from chicken.

We have cloned a novel matrix metalloproteinase (MMP) from cultured chicken embryo fibroblasts. The cDNA-derived protein sequence contains 608 amino acids including a C-terminal hydrophobic transmembrane domain of 24 amino acids and a cytoplasmic domain of 20 amino acids. This chicken MMP is 72% similar to a recently described membrane-type MMP (MT-MMP) from human placenta (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994) Nature 370, 61-65). Accordingly, we name this novel MMP chicken MT-MMP. As shown by Northern blotting, two MT-MMP mRNAs of 6 and 10 kilobases are constitutively expressed but only modestly regulated by growth factors and cytokines in cultured chicken embryo fibroblasts. Both mRNAs are abundant in the head and body of 8- and 9-day-old chicken embryos. As shown by in situ mRNA hybridization, MT-MMP is expressed in embryonic neural tube, spinal ganglia, and respiratory epithelium, as well as in developing cartilage and muscle. Using reverse transcription-polymerase chain reaction, we have found MT-MMP mRNA in 2-day-old chicken embryos and extraembryonic membranes. In addition, a strong correlation was observed between the mRNA expression of MT-MMP and 72-kDa type IV collagenase. Collectively, the early MT-MMP mRNA expression and its co-localization in several tissues with 72-kDa type IV collagenase mRNA suggest that the MT-MMP plays an important role in early development.

Progression of coronary atherosclerosis is associated with a common genetic variant of the human stromelysin-1 promoter which results in reduced gene expression.

There is a common polymorphism in the promoter sequence of the human stromelysin-1 gene, with one allele having a run of six adenosines (6A) and the other five adenosines (5A). We have previously reported, in a 3-year follow-up study of patients with coronary atherosclerosis, that those patients who are homozygous for the 6A allele show a more rapid progression of the disease. In this study, we have investigated whether the 5A/6A promoter polymorphism plays a role in the regulation of stromelysin-1 gene expression. In transient transfection experiments, a stromelysin-1 promoter construct with 6A at the polymorphic site was found to express less of the chloramphenicol acetyltransferase reporter gene than a construct containing 5A. Electrophoretic mobility shift assay and DNase I footprinting revealed the interaction of one or more nuclear protein(s) with the DNA sequence at the 5A/6A polymorphic site. The binding of one of the nucleoprotein factors was more readily detectable with an oligonucleotide probe corresponding to the 6A allele as compared with a probe corresponding to the 5A allele. Replacing the core binding sequence with a random DNA sequence abolished the interaction between the nuclear protein(s) and the probe and also increased reporter gene expression in transiently transfected cells. Thus, the common 5A/6A polymorphism of the human stromelysin-1 promoter appears to play an important role in regulating stromelysin-1 gene expression and may be involved in the progression of coronary heart disease.

Characterization of the 46-kDa intermediates of matrix metalloproteinase 3 (stromelysin 1) obtained by site-directed mutation of phenylalanine 83.

The precursor of matrix metalloproteinase 3 (MMP-3/ stromelysin 1) is activated in vitro by proteinases or mercurial compounds by stepwise processes which include the initial formation of short-lived intermediates and the subsequent intermolecular cleavage of the His82-Phe83 bond to generate the fully activated mature MMP-3 (Nagase, H., Enghild, J. J., Suzuki, K., and Salvesen, G. (1990) Biochemistry 29, 5783-5789). To study the enzymatic properties of the intermediates we have mutated either His82 or Phe83 to Arg to obtain a stable MMP-3 intermediate. The mutant proteins were expressed in Chinese hamster ovary K-1 cells using a mammalian expression system. The proMMP-3(H82R) mutant was activated by chymotrypsin, elastase, and 4-aminophenylmercuric acetate to the 45-kDa MMP-3 with similar mechanism and kinetics as the wild-type. In contrast, the activation of the proMMP-3(F83R) mutant by proteinases or 4-aminophenylmercuric acetate resulted in 46-kDa forms, which retained 13, 14, or 15 amino acids of the pro-domain depending on the activators. The proteinase-activated MMP-3(F83R) intermediates exhibited little enzymatic activity, but they were partially active after treatment with SH-reacting reagents. These molecules could bind to the tissue inhibitor of metalloproteinases-1 and alpha 2-macroglobulin. However, the SH group of Cys75 of the intermediates was not modified by SH-reagents, indicating that the enzymatic activity generated by SH-reagents resulted from molecular perturbation of the enzyme rather than their interaction with Cys75. When gelatin and transferrin were digested with the 46-kDa intermediates the products were different from those generated by the wild-type MMP-3, suggesting an alteration in substrate specificity. The treatment of proMMP-3 with trypsin resulted in the formation of a 45-kDa MMP-3 with an NH2-terminal Thr85, whose activity and substrate specificity were similar to those of the 46-kDa MMp-3(F83R) obtained from the proMMP-3(F83R) mutant. These observations indicate that the correct processing at the His82-Phe83 bond is critical for expression of the full activity and the specificity of MMP-3.

Promoter elements in the transcriptional activation of the human stromelysin-1 gene by the inflammatory cytokine, interleukin 1.

Stromelysin-1, a tissue-remodelling metalloproteinase synthesized by fibroblasts, has proteolytic activity against a variety of extracellular matrix components. Stromelysin-1 gene transcription is induced by the inflammatory cytokine interleukin (IL)-1. In fibroblasts transiently transfected with constructs containing 5'-deletion mutants of the human stromelysin-1 gene promoter, IL-1-induced transcriptional activity was abolished with the removal of region -102 to -54. This region includes an AP-1 binding site at positions -70 to -64. The AP-1 site alone increased the basal activity of and conferred minimal IL-1 inducibility onto the heterologous gene promoter of thymidine kinase. Interestingly, although the removal of the AP-1 site from the native promoter (-1303 to +4) affected the absolute levels of IL-1-induced and basal promoter activity, it did not alter their ratio, indicating the involvement of regions outside the AP-1 site in the IL-1 response. Of the stromelysin-1 5' flanking sequence examined, only the region -274 to -54 could confer IL-1 inducibility to a heterologous promoter independently of the AP-1 site. This region also bound specific nuclear factors. Further analysis revealed that the region composed of -86 to -71 and -63 to -54 could independently respond to IL-1 and bind protein of whole cell extracts. Protein binding to this region and to the AP-1 site was modestly induced by IL-1 treatment. From these results we conclude that, in fibroblasts, the AP-1 site (-70 to -64) is not necessary for the IL-1 response; however, it probably interacts through protein associations with the responsive region immediately surrounding it in the absolute transcriptional activation of the human stromelysin-1 gene by IL-1.

Different mechanisms of regulation of the human stromelysin and collagenase genes. Analysis by a reverse-transcription-coupled-PCR assay.

Tissue-remodeling processes are largely controlled by matrix metalloproteinases that degrade the extracellular components of connective tissues. In this study, gene regulation of two human matrix metalloproteinases, stromelysin and collagenase, was investigated by a reverse-transcription-coupled (RT)-PCR assay. Here, signals from both the heterogenous nuclear RNA (hnRNA) and mRNA are amplified, allowing the regulation of gene expression to be divided between transcriptional and/or post-transcriptional control. In confluent human lung fibroblast cultures, tumor-necrosis factor-alpha and 12-O-tetradecanoyl-phorbol 13-acetate induce stromelysin and collagenase genes transcriptionally. Interleukin-1 beta (IL-1 beta) induces stromelysin gene transcription but has little, if any, effect on the collagenase gene transcription in cells cultured in the presence of 10% serum. By a competitive RT-PCR assay, the IL-1 beta-reated cultures contain an average of 60 molecules of stromelysin mRNA/cell and the untreated cultures about 1.9 molecules/cell. In serum-starved cells, both IL-1 beta and serum induce transcription of the collagenase gene. Also, in serum-starved cells type II collagen can induce collagenase mRNA but not stromelysin mRNA. Inhibition of protein synthesis with cycloheximide induces stromelysin gene transcription but has no effect on the collagenase gene. These data indicate different mechanisms of regulation of the human stromelysin and collagenase genes in cultured cells.

Uncoordinate regulation of collagenase, stromelysin, and tissue inhibitor of metalloproteinases genes by prostaglandin E2: selective enhancement of collagenase gene expression in human dermal fibroblasts in culture.

The degradative effects of interleukin-1 (IL-1) on the extracellular matrix of connective tissue are mediated primarily by metalloproteinases and prostaglandins. Clinical observations suggest that these effects can be prevented, to some extent, by the use of non-steroidal anti-inflammatory drugs. We have examined the role of prostaglandin E2 (PGE2) in IL-1-induced gene expression by human skin fibroblasts in culture. Incubation of confluent fibroblast cultures with varying concentrations (0.01-1.0 microgram/ml) of PGE2 led to a dose-dependent elevation of collagenase mRNA steady-state levels, the promoter activity, and the secretion of the protein, whereas relatively little effect was observed on stromelysin and TIMP gene expression. Exogenous PGE2 had no additive or synergistic effect with IL-1 on collagenase gene expression. Furthermore, commonly used non-steroidal anti-inflammatory drugs (indomethacin, acetyl salicylic acid and ibuprofen), at doses which block prostaglandin synthesis in cultured fibroblasts, failed to counteract IL-1-induced collagenase and stromelysin gene expression, nor did they affect TIMP expression. Although the effects of PGE2 did not potentiate those of IL-1 on collagenase gene expression in vitro, one could speculate that massive production of PGE2 by connective tissue cells in vivo in response to inflammatory mediators such as IL-1 or tumor necrosis factor-alpha, could lead to sustained expression of collagenase in connective tissue cells after clearance of the growth factors.

A polyomavirus enhancer A-binding protein-3 site and Ets-2 protein have a major role in the 12-O-tetradecanoylphorbol-13-acetate response of the human stromelysin gene.

The expression of stromelysin, a major matrix metalloproteinase of connective tissues, is regulated by several cytokines, growth factors, protooncogenes as well as by 12-O-tetradecanoylphorbol-13-acetate (TPA). The human stromelysin gene promoter contains an activator protein-1 (Fos/Jun) binding site at -70, which is required for basal expression but is not necessary for the TPA response. In this study, using promoter deletion mutants in transient gene transfection experiments, we first identify the sequence from -220 to -202 as necessary for the TPA response of the stromelysin gene. Further, among the restriction fragments from the 1.3-kilobase long promoter, only the proximal fragment (-274 to -101) conferred a TPA response on the heterologous thymidine kinase gene promoter. The -220 to -202 sequence contains two copies of a motif similar to the polyomavirus enhancer A-binding protein-3 (PEA-3) site, which binds the Ets family of oncoproteins and transcription factors. Point mutations of either one of the two PEA-3 sites, in the 1.3-kilobase long stromelysin promoter context, reduced basal gene expression. However, only the mutation of the proximal, but not the distal PEA-3 site, significantly inhibited the TPA response. In cotransfection experiments, the Ets-2 protein transactivated the stromelysin promoter and the promoter proximal fragment containing the PEA-3 sites but not the promoters containing mutated PEA-3 sites. These data suggest that the PEA-3 site, but not the activator protein-1 site, and Ets-2 protein have a major role in the TPA induction of the human stromelysin gene transcription.

An enhancer for transcription of collagen IV genes is activated by F9 cell differentiation.

Expression of collagen IV genes is developmentally regulated and cell type-specific. To identify transcriptional control elements for the mouse alpha 2(IV) collagen gene, several promoter constructs were transiently transfected into mouse PYS-2 (parietal yolk sac) cells. Within the 5.5-kb (kilobase) upstream and 8.5-kb downstream sequences from the transcription start site, we have identified several regulatory active regions. Here, we report characterization of the most proximal 0.3-kb enhancer found at 4.5-kb upstream of the alpha 2(IV) collagen gene. This enhancer is transcriptionally active in cells that make collagen IV such as PYS-2 cells and differentiated F9 cells, but has little if any activity in cells that do not make collagen IV including NIH 3T3 cells and undifferentiated F9 embryonal carcinoma stem cells. This enhancer, linked to the herpes simplex virus TK gene promoter, confers a cell type-specific and differentiation-induced expression on the TK gene as well. Mutational and 5' and 3' deletion analysis demonstrate that this enhancer activity requires two identical response elements (GAACAAT) present in the 0.3-kb enhancer sequence. In gel shift assay, the GAACAAT element forms a complex that is specific for cells that make collagen IV.