Epigenetic regulation of mmp-9 gene expression

Matrix metalloproteinase 9 (MMP-9) is one of the most studied enzymes in cancer. MMP-9 can cleave proteins of the extracellular matrix and a large number of receptors and growth factors. Accordingly, its expression must be tightly regulated to avoid excessive enzymatic activity, which is associated with disease progression. Although we know that epigenetic mechanisms play a central role in controlling mmp-9 gene expression, predicting how epigenetic drugs could be used to suppress mmp-9 gene expression is not trivial because epigenetic drugs also regulate the expression of key proteins that can tip the balance towards activation or suppression of MMP-9. Here, we review how our understanding of the biology and expression of MMP-9 could be exploited to augment clinical benefits, most notably in terms of the prevention and management of degenerative diseases and cancer.     

Antitumoral effects of 9-cis retinoic acid in adrenocortical cancer

The currently available medical treatment options of adrenocortical cancer (ACC) are limited. In our previous meta-analysis of adrenocortical tumor genomics data, ACC was associated with reduced retinoic acid production and retinoid X receptor-mediated signaling. Our objective has been to study the potential antitumoral effects of 9-cis retinoic acid (9-cisRA) on the ACC cell line NCI-H295R and in a xenograft model. Cell proliferation, hormone secretion, and gene expression have been studied in the NCI-H295R cell line. A complex bioinformatics approach involving pathway and network analysis has been performed. Selected genes have been validated by real-time qRT-PCR. Athymic nude mice xenografted with NCI-H295R have been used in a pilot in vivo xenograft model. 9-cisRA significantly decreased cell viability and steroid hormone secretion in a concentration- and time-dependent manner in the NCI-H295R cell line. Four major molecular pathways have been identified by the analysis of gene expression data. Ten genes have been successfully validated involved in: (1) steroid hormone secretion (HSD3B1, HSD3B2), (2) retinoic acid signaling (ABCA1, ABCG1, HMGCR), (3) cell-cycle damage (GADD45A, CCNE2, UHRF1), and the (4) immune response (MAP2K6, IL1R2). 9-cisRA appears to directly regulate the cell cycle by network analysis. 9-cisRA also reduced tumor growth in the in vivo xenograft model. In conclusion, 9-cisRA might represent a promising new candidate in the treatment of hormone-secreting adrenal tumors and adrenocortical cancer.     

Production of insulin-like growth factor I (IGF-I) and IGF-binding proteins by rat intestinal stromal cells in vitro

To determine if intestinal stromal cells secrete diffusible factors such as insulin-like growth factors (IGFs) capable of regulating epithelial cell growth in vitro, stromal cells were isolated by enzymatic digestion of rat intestine. Incorporation of [³H]thymidine into DNA and [¹⁴C]leucine into protein of IEC-6 cells, a model intestinal epithelial cell line, was significantly increased (two- to threefold) when the IEC-6 cells were co-cultured with stromal cells, relative to IEC-6 cells grown alone. Medium conditioned by stromal cells stimulated DNA synthesis of IEC-6 cells in a dose-dependent manner. Analysis of the conditioned medium revealed that intestinal stromal cells secreted IGF-I, but little IGF-II, in addition to an M _r 32,000 IGF-binding protein (IGFBP-2) and an IGFBP having M _r∼ 24,000. We conclude that rat intestinal stromal cells secrete one or more diffusible factors, which may include IGF-I and IGFBPs, capable of stimulating proliferation of IEC-6 cells in vitro. Received 25 August 1997; received after revision 7 November 1997; accepted 20 November 1997     

Muscle LIM protein promotes expression of the acetylcholine receptor <Emphasis Type="Italic">γ</Emphasis>-subunit gene cooperatively with the myogenin-E12 complex

Muscle LIM protein (MLP, also referred to as CRP3) is a muscle-specific LIM-only protein, which consists of two LIM motifs. MLP functions as a positive regulator during myogenesis. Here we report that MLP serves as a cofactor regulating the expression of the nicotinic acetylcholine receptor (AChR) -subunit gene in skeletal muscle cells. We found that MLP promoted the expression of the AChR -subunit gene in C2C12 myotubes, but not in C2C12 myoblasts or NIH3T3 fibroblasts. Furthermore, we showed that MLP interacted with myogenin in vivo and enhanced the binding ability of the myogenin-E12 heterodimer to the E boxes in the AChR -subunit gene promoter. Together, these results suggest that MLP promotes the specific expression of the AChR -subunit gene cooperatively with the myogenin-E12 complex during myogenesis.Received 17 May 2004; received after revision 22 July 2004; accepted 26 July 2004     

Knockout of the Bcmo1 gene results in an inflammatory response in female lung, which is suppressed by dietary beta-carotene

Beta-carotene 15,15′-monooxygenase 1 knockout (Bcmo1 ^?/?) mice accumulate beta-carotene (BC) similarly to humans, whereas wild-type (Bcmo1 ^+/+) mice efficiently cleave BC. Bcmo1 ^?/? mice are therefore suitable to investigate BC-induced alterations in gene expression in lung, assessed by microarray analysis. Bcmo1 ^?/? mice receiving control diet had increased expression of inflammatory genes as compared to BC-supplemented Bcmo1 ^?/? mice and Bcmo1 ^+/+ mice that received either control or BC-supplemented diets. Differential gene expression in Bcmo1 ^?/? mice was confirmed by real-time quantitative PCR. Histochemical analysis indeed showed an increase in inflammatory cells in lungs of control Bcmo1 ^?/? mice. Supported by metabolite and gene-expression data, we hypothesize that the increased inflammatory response is due to an altered BC metabolism, resulting in an increased vitamin A requirement in Bcmo1 ^?/? mice. This suggests that effects of BC may depend on inter-individual variations in BC-metabolizing enzymes, such as the frequently occurring human polymorphisms in BCMO1.     

Binding of matrilysin-1 to human epithelial cells promotes its activity

Matrix metalloproteinase-7 (MMP-7, matrilysin- 1) modulates crucial biological events by processing many epithelial cell surface-associated effectors. We addressed MMP-7 interaction with human epithelial cells and its resulting activity. In human endometrium, a model of controlled tissue remodeling, proMMP-7 was diffusely immunolocalized inside epithelial cells, whereas MMP-7 delineated their entire plasma membrane. Endometrial explants preferentially retained active MMP-7, but not proMMP-7. Endometrial epithelial cells and carcinoma cells from various tissues bound active MMP-7. Endometrial carcinoma-derived Ishikawa cells showed high affinity (K_D of ~2.5 nM) and capacity (~260 000 sites per cell) for MMP-7. MMP-7 binding decreased by extracting membrane sterols or interfering with heparan sulfate proteoglycans, and was abrogated by tissue inhibitors of metalloproteinase-2 (TIMP-2) or synthetic MMP inhibitors. Bound MMP-7 not only remained fully active towards a macromolecular substrate but also became resistant to TIMP-2. We conclude that MMP-7-selective targeting to the plasma membrane of epithelial cells promotes its activity by conferring resistance to TIMP-2. A. Berton, C. Selvais: These authors contributed equally to this work. P. J. Courtoy, E. Marbaix, H. Emonard: These authors contributed equally to the supervision of this work. Received 20 September 2006; received after revision 30 November 2006; accepted 18 January 2007     

Expression ofPseudomonas phosphotriesterase activity in the fall armyworm confers resistance to insecticides

Summary The gene encoding for the phosphotriesterase (opd) fromPseudomonas diminuta has been subcloned into a baculovirus expression system. Functional enzyme is produced when the recombinant baculovirus is used to infect either culturedSpodoptera frugiperda sf9 cells or the larval stage of the fall armyworm. The LD₅₀ for paraoxon toxicity was found to increase 280-fold in the larvae after infection with the recombinant baculovirus and expression of the functional phosphotriesterase.This work was supported by the Army Research Office (DAAL03-87-K-0017) and the Texas Advanced Technology Program. F.M.R. is the recipient of NIH Research Career Development Award DK-01366.     

The protease-activated receptor-3 (PAR-3) can signal autonomously to induce interleukin-8 release

Protease-activated receptors (PARs) play a clear role in the burst of inflammatory reactions and immune responses. However, for PAR-3, the most elusive member of the PAR family, the functional role is still largely unclear. It has been claimed that PAR-3 does not signal autonomously, although the wide expression of human PAR-3 indicates its important physiological roles. We demonstrate that in HEK-293 cells, stably transfected with human PAR-3, thrombin induced calcium signaling, IL-8 gene expression and IL-8 release. We confirmed this finding using human lung epithelial and human astrocytoma cells that express endogenous PAR-3. Moreover, thrombin exposure of HEK-293 cells resulted in ERK1/2 activation coinciding with IL-8 release. The effects of thrombin were not dependent on PAR-1 activation, as confirmed by PAR-1 gene silencing. Thus, we propose that PAR-3 is able to signal autonomously to induce IL-8 release mediated by ERK1/2 phosphorylation, which contributes actively to inflammatory responses. Received 9 December 2007; received after revision 16 January 2008; accepted 18 January 2008     

Recombinant synthesis of mouse Zn3-β and Zn4-α metallothionein 1 domains and characterization of their cadmium(II) binding capacity

Genetic engineering, coupled with spectro scopic analyses, has enabled the metal binding proper ties of the α and β subunits of mouse metallothionein 1 (MT) to be characterized. A heterologous expression system in E.coli has led to high yields of their pure zinc-complexed forms. The cadmium(II) binding properties of recombinant Zn₄-αMT and Zn₃-βMT have been studied by electronic absorption and circular dichroism. The former binds Cd(II) identically to α fragments obtained from mammalian organs, showing that the recombinant polypeptide behaves like the na tive protein. Titration of Zn₃-βMT with CdCl₂ results in the formation of Cd₃-βMT. The addition of excess Cd(II) leads to Cd₄-βMT which, with the extra loading of Cd(II), unravels to give rise isodichroically to Cd₉-βMT. The effect of cadmium-displaced Zn(II) ions and excess Cd(II) above the full metal occupancy of three has been studied using Chelex-100. The Cd₃-βMT species is stable in the presence of this strong metal-chelating agent. Received 20 May 1997; received after revision 7 July 1997; accepted 9 July 1997     

Endocrine-dependent expression of circadian clock genes in insects

Current models state that insect peripheral oscillators are directly responsive to light, while mammalian peripheral clock genes are coordinated by a master clock in the brain via intermediate factors, possibly hormonal. We show that the expression levels of two circadian clock genes, period (per) and Par Domain Protein 1 (Pdp1) in the peripheral tissue of an insect model species, the linden bug Pyrrhocoris apterus, are inversely affected by contrasting photoperiods. The effect of photoperiod on per and Pdp1 mRNA levels was found to be mediated by the corpus allatum, an endocrine gland producing juvenile hormone. Our results provide the first experimental evidence for the effect of an endocrine gland on circadian clock gene expression in insects. Received 31 October 2007; received after revision 7 January 2008; accepted 9 January 2008 D. Dolezel, L. Zdechovanova: These authors contributed equally to this work.     

Multiple roles of the DSCR1 (Adapt78 or RCAN1) gene and its protein product Calcipressin 1 (or RCAN1) in disease

The DSCR1 (Adapt78) gene¹ is transiently induced by stresses to temporarily protect cells against further potentially lethal challenges. However, chronic expression of the DSCR1 (Adapt78) gene has now been implicated in several pathological conditions including Alzheimer’s disease, Down syndrome and cardiac hypertrophy. Calcipressin 1 has been shown to function through direct binding and inhibition of the serine threonine protein phosphatase Calcineurin. Pharmacological inhibition of calcineurin, by the immunosuppressive drugs cyclosporin A and FK506, affects a wide variety of diseases. It is, therefore, likely that this endogenous calcineurin inhibitor, calcipressin 1, may also play a role in a variety of human diseases. ¹Please note that the mammalian DSCR1 gene is also called Adapt78 or RCAN1, and its protein products have been named Calcipressin1, MCIP1 and RCAN1. A proposal to adopt a single gene name of RCAN1 and a protein name RCAN1 (for Regulator of Calcineurin) has been endorsed by the HUGO Gene Nomenclature Committee, but final approval must await agreement from a majority of researchers in the field. Received 2 March 2005; received after revision 27 May 2005; accepted 19 July 2005