Dissection Analysis of the Negative Regulation of the Human Urokinase-type Plasminogen Activator (uPA) Gene

The urokinase-type plasminogen activator (uPA) is a serine protease involved in processes such as cell migration and invasion through the activation of the extracellular protease plasmin responsible of the degradation of the proteins of the extracellular matrix. The ability of the cells to migrate and to invade the surrounding tissues plays a fundamental role in many normal and pathological processes such as fibrinolysis, wound healing, angiogenesis, embryogenesis, gametogenesis, ovulation, mammary gland involution and tumor metastasis. Due to its high destructive potential, localized activation of plasminogen is regulated by a complex network of molecular interactions involving both specific inhibitors (PAI-1 and PAI-2), and cell bound receptor (uPAR) and the synthesis of every component of the plasminogen activator system is tightly regulated by a number of factors like hormones, growth factors and cytokines. On the other hand, the restricted expression of uPA in the organism to a very few cell types (kidney and lung), its inducibility by different stimuli and its overexpression in tumours and several transformed and tumoral cell lines, indicate that uPA regulation occurs also at the level of gene expression. The gene encoding uPA and its 5’ flanking region have been sequenced and characterized in human, mouse and pig. Both in vivo and in vitro studies of progressive 5'deletions of the regulatory region of uPA, have revealed the presence of positive and negative cis-acting sequences and of specific contributions of the proximal regulatory regions to cell-type specific expression of the gene, suggesting the presence of multiple array of cis-acting sequences specific for different transcription factors that direct uPA expression in a celltype specific manner. In the human uPA promoter the enhancer is located between -2100 and -1870 from the transcriptional start site and contains two binding sites for the transcription factor API and PE A3 which are important for both the constitutive and regulated expression of the gene. Furthermore, the cooperation between the two API sites is required for TPA inducibility and is mediated by other proteins called Upstream Enhancer Factors (UEF). A negative cis-acting element has been localized between -1870 and -1570 and contains a binding site for a multiproteic complex (NF-kB/c-rel) that mediates the inducibility by TPA in HeLa and HepG2 cell lines. Much evidence, however, indicates that the regulation of uPA is mainly negative. The studies described in this thesis have led to the identification of negative regulatory elements that may play an important role in the establishment of a silenced phenotype in a cell-type specific manner. The analysis of promoter activity in cell lines not expressing (HeLa and CVl) and expressing uPA (PC3) allowed the identification of at least three regions that play different roles in the silencing of the uPA gene in different cell lines, named SI (-1870/-1428), 82 (-787/-537) and S3 (-537/-86). Of the three only S2 shows cell-type specificity, as it is active only in cells that do not express uPA; S1 and S3, on the other hand, could act as modulators of uPA gene expression in those cells that express uPA. All of them negatively regulate the activity of the minimal promoter, suggesting that they can interfere with the formation of a competent pre-initiation complex at the start site of transcription. A dissection analysis of silencer S2 detected the presence of multiple silencing units, although the deletion of a single one does not have any effect on the activity of the promoter. Transcription from a heterologous promoter is affected only when more than one copy of a single unit is cloned in front of it, suggesting that in the context of the whole S2 region each unit acts synergistically with the others in the silencing of the gene. DNase I footprinting analysis of S2 showed that this region is extensively protected by nuclear extract from uPA producing (PC-3) and not producing (HeLa) cell lines, with few differences between them. However, the LMSA analysis of the complexes indicated that their molecular composition may be different in the two cell lines tested. In particular, in HeLa cell extract, a single strand binding activity seems to bind S2 and it may be responsible for the assembly of a negative-acting complex that would prevent the loading of a competent transcription initiation complex at the start site of transcription. Furthermore, the presence of two binding sites for proteins belonging to the HMG-box containing protein family, suggested that the assembly of a stereospecific complex is required for the activity of silencer S2, although it is not clear if their activity is required for the silencing of the gene or for the inactivation of the inhibition in those cells that express uPA as in HepG2 cell line. In these cells uPA is expressed at a very low basal level, but its expression is inducible by TPA. In this cell line S2 is not active; on the contrary its presence is required for basal expression of uPA. The presence in S2 of a sequence matching the transforming growth factor β inihibitory element (TIE), described to be important for mediating the inhibition of TPA induction by TGF-βl of the stromelysin gene, has led me to investigate the role of this element on S2 activity in HepG2 cells. Transient transfection analysis of uPA promoter deletions showed that, at least in this system, the TIE is not involved in the regulation of uPA expression. Preliminary results, shown in appendix 2, have suggested another interesting aspect of the negative regulation of uPA expression by the p53 tumor suppressor gene product, although no p53 binding to uPA promoter has been shown so far. In conclusion, the studies presented in this thesis have showed that a complex array of regulatory sequences, in addition to the enhancer and minimal promoter, appear to regulate uPA transcription, some of which are cell-type specific

PHOX2A and PHOX2B are differentially regulated during retinoic acid-driven differentiation of SK-N-BE(2)C neuroblastoma cell line

AbstractPHOX2B and its paralogue gene PHOX2A are two homeodomain proteins in the network regulating the development of autonomic ganglia that have been associated with the pathogenesis of neuroblastoma (NB), because of their over-expression in different NB cell lines and tumour samples. We used the SK-N-BE(2)C cell line to show that all-trans retinoic acid (ATRA), a drug that is widely used to inhibit growth and induce differentiation in NBs, regulates both PHOX2A and PHOX2B expression, albeit by means of different mechanisms: it up-regulates PHOX2A and down-regulates PHOX2B. Both mechanisms act at transcriptional level, but prolonged ATRA treatment selectively degrades the PHOX2A protein, whereas the corresponding mRNA remains up-regulated. Further, we show that PHOX2A is capable of modulating PHOX2B expression, but this mechanism is not involved in the PHOX2B down-regulation induced by retinoic acid. Our findings demonstrate that PHOX2A expression is finely controlled during retinoic acid differentiation and this, together with PHOX2B down-regulation, reinforces the idea that they may be useful biomarkers for NB staging, prognosis and treatment decision making

Special Issue: Cholinergic Control of Inflammation

Inflammation caused by infection, tissue trauma, and disease states such as arthritis and inflammatory bowel disease is perceived by the Central nervous System (CNS) through different routes that, by means of neural reflex circuits, regulate the immune system response [...

Unanswered questions in the regulation and function of the duplicated α7 nicotinic receptor gene CHRFAM7A

The α7 nicotinic receptor (α7 nAChR) is an important entry point for Ca2+ into the cell, which has broad and important effects on gene expression and function. The gene (CHRNA7), mapping to chromosome (15q14), has been genetically linked to a large number of diseases, many of which involve defects in cognition. While numerous mutations in CHRNA7 are associated with mental illness and inflammation, an important control point may be the function of a recently discovered partial duplication CHRNA7, CHRFAM7A, that negatively regulates the function of the α7 receptor, through the formation of heteropentamers; other functions cannot be excluded. The deregulation of this human specific gene (CHRFAM7A) has been linked to neurodevelopmental, neurodegenerative, and inflammatory disorders and has important copy number variations. Much effort is being made to understand its function and regulation both in healthy and pathological conditions. However, many questions remain to be answered regarding its functional role, its regulation, and its role in the etiogenesis of neurological and inflammatory disorders. Missing knowledge on the pharmacology of the heteroreceptor has limited the discovery of new molecules capable of modulating its activity. Here we review the state of the art on the role of CHRFAM7A, highlighting unanswered questions to be addressed. A possible therapeutic approach based on genome editing protocols is also discussed

The Human-Restricted Isoform of the α7 nAChR, CHRFAM7A: A Double-Edged Sword in Neurological and Inflammatory Disorders

CHRFAM7A is a relatively recent and exclusively human gene arising from the partial duplication of exons 5 to 10 of the α7 neuronal nicotinic acetylcholine receptor subunit (α7 nAChR) encoding gene, CHRNA7. CHRNA7 is related to several disorders that involve cognitive deficits, including neuropsychiatric, neurodegenerative, and inflammatory disorders. In extra-neuronal tissues, α7nAChR plays an important role in proliferation, differentiation, migration, adhesion, cell contact, apoptosis, angiogenesis, and tumor progression, as well as in the modulation of the inflammatory response through the “cholinergic anti-inflammatory pathway”. CHRFAM7A translates the dupα7 protein in a multitude of cell lines and heterologous systems, while maintaining processing and trafficking that are very similar to the full-length form. It does not form functional ion channel receptors alone. In the presence of CHRNA7 gene products, dupα7 can assemble and form heteromeric receptors that, in order to be functional, should include at least two α7 subunits to form the agonist binding site. When incorporated into the receptor, in vitro and in vivo data showed that dupα7 negatively modulated α7 activity, probably due to a reduction in the number of ACh binding sites. Very recent data in the literature report that the presence of the duplicated gene may be responsible for the translational gap in several human diseases. Here, we will review the studies that have been conducted on CHRFAM7A in different pathologies, with the intent of providing evidence regarding when and how the expression of this duplicated gene may be beneficial or detrimental in the pathogenesis, and eventually in the therapeutic response, to CHRNA7-related neurological and non-neurological diseases

Etonogestrel Administration Reduces the Expression of PHOX2B and Its Target Genes in the Solitary Tract Nucleus

Heterozygous mutations of the transcription factor PHOX2B are responsible for Congenital Central Hypoventilation Syndrome, a neurological disorder characterized by inadequate respiratory response to hypercapnia and life-threatening hypoventilation during sleep. Although no cure is currently available, it was suggested that a potent progestin drug provides partial recovery of chemoreflex response. Previous in vitro data show a direct molecular link between progestins and PHOX2B expression. However, the mechanism through which these drugs ameliorate breathing in vivo remains unknown. Here, we investigated the effects of chronic administration of the potent progestin drug Etonogestrel (ETO) on respiratory function and transcriptional activity in adult female rats. We assessed respiratory function with whole-body plethysmography and measured genomic changes in brain regions important for respiratory control. Our results show that ETO reduced metabolic activity, leading to an enhanced chemoreflex response and concurrent increased breathing cycle variability at rest. Furthermore, ETO-treated brains showed reduced mRNA and protein expression of PHOX2B and its target genes selectively in the dorsal vagal complex, while other areas were unaffected. Histological analysis suggests that changes occurred in the solitary tract nucleus (NTS). Thus, we propose that the NTS, rich in both progesterone receptors and PHOX2B, is a good candidate for ETO-induced respiratory modulation

Effect of eNOS silencing on HIF-1α accumulation, VEGF secretion, mtDNA and ATP levels.

<p>(A) Characterization of HUVECs transfected with eNOS siRNA: densitometric analysis of eNOS protein expression where eNOS protein levels were normalized to β-actin protein. ***p<0.001; <i>t</i> test; n = 4. Inset: representative blots of eNOS protein in cells transfected with control (ctrl) or eNOS siRNA. (B) HUVECs were transfected with control (lane 2) or eNOS siRNA (lane 3), and HIF-1α protein was detected by western blotting on the corresponding nuclear extracts. In lane 1, nuclear extracts from untransfected cells. An aliquot of total cell lysates was immunoblotted with anti eNOS antibodies to check silencing, and with anti β-actin antibodies as loading control. A representative blot of 2 comparable experiments is shown. (C) VEGF protein levels were detected by ELISA measurement in conditioned media collected from HUVECs 48 h after transfection with control or eNOS siRNA. Results are expressed as pg of VEGF normalized to the cell protein content (pg/mg protein). *p<0.05; <i>t</i> test; n = 3. (D) MtDNA (left axis) and total cellular ATP content (right axis) were measured in HUVECs transfected for 48 h with control or eNOS siRNA. In silenced cells, mtDNA and ATP were reduced by 36±0.4 and 45±9.7% respectively. **p<0.01 and ***p<0.001; <i>t</i> test; n = 3.</p

The enhancement in HUVEC migration induced by L-NAME is reverted by the NO donor DETA-NO and is independent of the cGMP pathway.

<p>(A) HUVECs were treated for 48 h with 5 mM L-NAME in the absence or in the presence of 500 nM DETA/NO for the last 24 h, as indicated. Chemotaxis experiments were then performed using 25 ng/ml VEGF as attractants. Results are expressed as the number of migrating cells. #p<0.001 <i>vs</i> basal migration in control cells (CTRL); §p<0.01 <i>vs</i> VEGF-induced migration in control cells; ***p<0.001 <i>vs</i> basal migration in L-NAME treated cells; °°°p<0.001 <i>vs</i> VEGF-induced migration in L-NAME treated cells; no significant differences between control and DETA/NO treated cells (One-way ANOVA with Bonferroni's test, n = 15). (B) HUVECs were treated for 48 h with 5 mM L-NAME or 1 µM ODQ, and chemotaxis experiments were performed as described in (A). Results are expressed as the number of migrating cells in the different experimental conditions. #p<0.001 <i>vs</i> basal migration in control cells (CTRL); §p<0.001 <i>vs</i> VEGF-induced migration in control cells; no significant differences between control and ODQ treated cells (One-way ANOVA with Bonferroni's test, n = 3). (C) cGMP accumulation in HUVECs treated for 48 h with L-NAME or ODQ was evaluated by EIA and expressed as pmol of cGMP normalized to the cell protein content (pmol/mg protein). ***p<0.001; One-way ANOVA with Bonferroni's test; n = 3.</p

Effects of L-NAME treatment on eNOS, VEGF and KDR expression.

<p>(A) Densitometric analysis of eNOS protein expression. ***p<0.001; <i>t</i> test; n = 11. Inset: a representative blot out of eleven is shown. Total eNOS protein was evaluated by western blotting on lysates prepared from control cells (lane 1) or from 48 h L-NAME treated cells (lane 2). β-actin was used as a loading control. (B) eNOS RNA levels were measured by RT-qPCR and normalized to the level of the housekeeping gene 18S. No significant differences between control and L-NAME treated cells (<i>t</i> test, n = 3). (C) VEGF and KDR RNA levels were measured by RT-qPCR and normalized to the level of the housekeeping gene 18S. **p<0.01 <i>vs</i> control cells (CTRL); <i>t</i> test; n = 6−4 for VEGF and KDR, respectively. (D) VEGF protein levels were detected by ELISA measurement in conditioned media collected from control or 48 h L-NAME treated cells. Results are expressed as pg of VEGF normalized to the cell protein content (pg/mg protein). **p<0.01; <i>t</i> test; n = 3. (E) KDR protein was visualized by western blot after immunoprecipitation with KDR antibodies of HUVEC lysates obtained from control (lane 1) or from 48 h L-NAME treated cells (lane 2). An aliquot of total cell lysates was immunoblotted with β-actin antibodies as a control (input). Shown is a representative blot of 2 comparable experiments. (F) Control cells (lanes 1 and 2) or 48 h L-NAME treated cells (lanes 3 and 4) were stimulated for 5 min with 25 ng/ml VEGF. Aliquots of cell lysates were separated by 10% SDS-PAGE and immunoblotted with the indicated antibodies. Actin was used as a loading control. Shown is a representative blot of 4 comparable experiments.</p