Comparison between the intermalleolar distance measured on the couch and on the floor in axial spondyloarthritis.

The aim of this study was to assess whether axial spondyloarthritis (axial SpA) patients' supine position on the couch (OC) or on the floor (OF) affects intermalleolar distance (IMD) measurement and its Bath Ankylosing Spondylitis Metrology Index (BASMI) scoring, using all three versions of BASMI index. OC- and OF-IMDs were obtained for 43 axial SpA patients (M:F = 19:24). Age, gender, height, weight, body mass index (BMI), disease type and disease duration were also collected. Statistical analyses and correlations were performed as appropriate. Mean IMD measurements obtained with individuals in the two distinct measuring positions were not significantly different in the patients studied. Furthermore, there was a significant correlation between OC-IMD and OF-IMD values. There was no significant relationship between IMD and patient age, gender, height, weight, BMI, or disease duration. However, looking at disease type, IMDs of patients with ankylosing spondylitis (AS) were ~30 % greater than those with psoriatic arthritis (PsA) in our study population (p < 0.05). There were no significant differences between the measured patient characteristics that accounted for the greater IMDs of those diagnosed with AS. IMD measurements and resultant BASMI scores were the same whether the patient was positioned OC or OF in our axial SpA cohort. Unexpectedly, IMD measurements were significantly greater (~30 %) in AS patients than in axial PsA patients.

Angiogenic functions of voltage-gated Na+ Channels in human endothelial cells: modulation of vascular endothelial growth factor (VEGF) signaling.

Voltage-gated sodium channel (VGSC) activity has previously been reported in endothelial cells (ECs). However, the exact isoforms of VGSCs present, their mode(s) of action, and potential role(s) in angiogenesis have not been investigated. The main aims of this study were to determine the role of VGSC activity in angiogenic functions and to elucidate the potentially associated signaling mechanisms using human umbilical vein endothelial cells (HUVECs) as a model system. Real-time PCR showed that the primary functional VGSC α- and ß-subunit isoforms in HUVECs were Nav1.5, Nav1.7, VGSCß1, and VGSCß3. Western blots verified that VGSCα proteins were expressed in HUVECs, and immunohistochemistry revealed VGSCα expression in mouse aortic ECs in vivo. Electrophysiological recordings showed that the channels were functional and suppressed by tetrodotoxin (TTX). VGSC activity modulated the following angiogenic properties of HUVECs: VEGF-induced proliferation or chemotaxis, tubular differentiation, and substrate adhesion. Interestingly, different aspects of angiogenesis were controlled by the different VGSC isoforms based on TTX sensitivity and effects of siRNA-mediated gene silencing. Additionally, we show for the first time that TTX-resistant (TTX-R) VGSCs (Nav1.5) potentiate VEGF-induced ERK1/2 activation through the PKCα-B-RAF signaling axis. We postulate that this potentiation occurs through modulation of VEGF-induced HUVEC depolarization and [Ca(2+)](i). We conclude that VGSCs regulate multiple angiogenic functions and VEGF signaling in HUVECs. Our results imply that targeting VGSC expression/activity could be a novel strategy for controlling angiogenesis.

Estrogen and non-genomic upregulation of voltage-gated Na(+) channel activity in MDA-MB-231 human breast cancer cells: role in adhesion.

External (but not internal) application of beta-estradiol (E2) increased the current amplitude of voltage-gated Na(+) channels (VGSCs) in MDA-MB-231 human breast cancer (BCa) cells. The G-protein activator GTP-gamma-S, by itself, also increased the VGSC current whilst the G-protein inhibitor GDP-beta-S decreased the effect of E2. Expression of GPR30 (a G-protein-coupled estrogen receptor) in MDA-MB-231 cells was confirmed by PCR, Western blot and immunocytochemistry. Importantly, G-1, a specific agonist for GPR30, also increased the VGSC current amplitude in a dose-dependent manner. Transfection and siRNA-silencing of GPR30 expression resulted in corresponding changes in GPR30 protein expression but only internally, and the response to E2 was not affected. The protein kinase A inhibitor, PKI, abolished the effect of E2, whilst forskolin, an adenylate cyclase activator, by itself, increased VGSC activity. On the other hand, pre-incubation of the MDA-MB-231 cells with brefeldin A (a trans-Golgi protein trafficking inhibitor) had no effect on the E2-induced increase in VGSC amplitude, indicating that such trafficking ('externalisation') of VGSC was not involved. Finally, acute application of E2 decreased cell adhesion whilst the specific VGSC blocker tetrodotoxin increased it. Co-application of E2 and tetrodotoxin inhibited the effect of E2 on cell adhesion, suggesting that the effect of E2 was mainly through VGSC activity. Pre-treatment of the cells with PKI abolished the effect of E2 on adhesion, consistent with the proposed role of PKA. Potential implications of the E2-induced non-genomic upregulation of VGSC activity for BCa progression are discussed.

A simple technique for the prediction of interacting proteins reveals a direct Brn-3a-androgen receptor interaction.

The formation of multiprotein complexes constitutes a key step in determining the function of any translated gene product. Thus, the elucidation of interacting partners for a protein of interest is of fundamental importance to cell biology. Here we describe a simple methodology for the prediction of novel interactors. We have applied this to the developmental transcription factor Brn-3a to predict and verify a novel interaction between Brn-3a and the androgen receptor (AR). We demonstrate that these transcription factors form complexes within the nucleus of ND7 neuroblastoma cells, while in vitro pull-down assays show direct association. As a functional consequence of the Brn-3a-AR interaction, the factors bind cooperatively to multiple elements within the promoter of the voltage-gated sodium channel, Nav1.7, leading to a synergistic increase in its expression. Thus, these data define AR as a direct Brn-3a interactor and verify a simple interacting protein prediction methodology that is likely to be useful for many other proteins.

Alternative splicing of Nav1.5: an electrophysiological comparison of 'neonatal' and 'adult' isoforms and critical involvement of a lysine residue.

In developmentally regulated D1:S3 splicing of Nav1.5, there are 31 nucleotide differences between the 5'-exon ('neonatal') and the 3'-exon ('adult') forms, resulting in 7 amino acid differences in D1:S3-S3/S4 linker. In particular, splicing replaces a conserved negative aspartate residue in the 'adult' with a positive lysine. Here, 'neonatal' and 'adult' Nav1.5 alpha-subunit splice variants were stably transfected into EBNA-293 cells and their electrophysiological properties investigated by whole-cell patch-clamp recording. Compared with the 'adult' isoform, the 'neonatal' channel exhibited (1) a depolarized threshold of activation and voltage at which the current peaked; (2) much slower kinetics of activation and inactivation; (3) 50% greater transient charge (Na(+)) influx; (4) a stronger voltage dependence of time to peak; and (5) a slower recovery from inactivation. Tetrodotoxin sensitivity and VGSCbeta1-4 mRNA expression levels did not change. The significance of the charge-reversing aspartate to lysine substitution was investigated by mutating the lysine in the 'neonatal' channel back to aspartate. In this 'neonatal K211D' mutant, the electrophysiological parameters studied strongly shifted back towards the 'adult', that is the lysine residue was primarily responsible for the electrophysiological effects of Nav1.5 D1:S3 splicing. Taken together, these data suggest that the charge reversal in 'neonatal' Nav1.5 would (1) modify the channel kinetics and (2) prolong the resultant current, allowing greater intracellular Na(+) influx. Developmental and pathophysiological consequences of such differences are discussed.

Cardiac expression of Brn-3a and Brn-3b POU transcription factors and regulation of Hsp27 gene expression.

The Brn-3 family of transcription factors play a critical role in regulating expression of genes that control cell fate, including the small heat shock protein Hsp27. The aim of this study was to investigate the relationship between Brn-3a and Brn-3b and Hsp27 expression in the developing rodent heart. Brn-3a and Brn-3b were detected from embryonic days 9.5-10.5 (E9.5-E10.5) in the mouse heart, with significant increases seen later during development. Two isoforms (long and short) of each protein were detected during embryogenesis and postnatally. Brn-3a messenger RNA (mRNA) and protein were localized by E13.0 to the atrio-ventricular (AV) valve cushions and leaflets, outflow tract (OFT), epicardium and cardiac ganglia. By E14.5, Brn-3a was also localised to the septa and compact ventricular myocardium. An increase in expression of the long Brn-3a(l) isoform between E17 and adult coincided with a decrease in expression of Brn-3b(l) and a marked increase in expression of Hsp27. Hearts from Brn-3a-/- mice displayed a partially penetrant phenotype marked by thickening of the endocardial cushions and AV valve leaflets and hypoplastic ventricular myocardium. Loss of Brn-3a was correlated with a compensatory increase in Brn-3b and GATA3 mRNA but no change in Hsp27 mRNA. Reporter assays in isolated cardiomyocytes demonstrated that both Brn-3a and Brn-3b activate the hsp27 promoter via a consensus Brn-3-binding site. Therefore, Brn-3 POU factors may play an important role in the development and maintenance of critical cell types and structures within the heart, in part via developmental regulation of myocardial Hsp27 expression. Furthermore, Brn-3a may be necessary for correct valve and myocardial remodelling and maturation.

Identification and characterization of the promoter region of the Nav1.7 voltage-gated sodium channel gene (SCN9A).

The Nav1.7 sodium channel plays an important role in pain and is also upregulated in prostate cancer. To investigate the mechanisms regulating physiological and pathophysiological Nav1.7 expression we identified the core promoter of this gene (SCN9A) in the human genome. In silico genomic analysis revealed a putative SCN9A 5' non-coding exon approximately 64,000 nucleotides from the translation start site, expression of which commenced at three very closely-positioned transcription initiation sites (TISs), as determined by 5' RACE experiments. The genomic region around these TISs possesses numerous core elements of a TATA-less promoter within a well-defined CpG island. Importantly, it acted as a promoter when inserted upstream of luciferase in a fusion construct. Moreover, the activity of the promoter-luciferase construct ostensibly paralleled endogenous Nav1.7 mRNA levels in vitro, with both increased in a quantitatively and qualitatively similar manner by numerous factors (including NGF, phorbol esters, retinoic acid, and Brn-3a transcription factor over-expression).

Post-transcriptional regulation of the Brn-3b transcription factor in differentiating neuroblastoma cells.

The post-transcriptional control of mRNA levels is a very powerful mechanism which allows cells to quickly change the amount of specific proteins. In this study, we wanted to analyze whether the Brn-3b transcription factor, essential for the proper development of mouse retinal ganglion cells, is subjected to such post-transcriptional regulation. In particular, due to its conservation amongst different species, we wanted to study the role of its 3' untranslated region (3'UTR). We show that the 3'UTR of the Brn-3b mRNA does indeed contain regulatory sequences that mediate mRNA degradation upon serum starvation-induced differentiation of ND7 neuroblastoma cells. The specific region mediating this effect has been characterized and two different microRNAs that potentially regulate the stability of Brn-3b have been identified. Moreover we show that Dicer, one of the key enzymes in the production of microRNAs, is strongly up-regulated in ND7 cells subjected to differentiation.

p300 activation by Presenilin 1 but not by its M146L mutant.

The transcriptional co-activator p300 plays an important role in regulating gene expression in a number of different cell types. We have shown that wild type (WT) Presenilin 1 (PS1) stimulates the transcriptional activity ability of CREB Binding Protein (CBP), a close homolog of p300, whereas the Alzheimer's disease (AD) associated mutant of PS1 does not have this effect. A recent report has suggested that mutant PS1 can also disrupt the TCF/beta-catenin/CBP interaction but has no effect on the TCF/beta-catenin/p300 interaction. This suggests that the malregulation of CBP, but not of p300, caused by mutation in PS1 may be involved in the disease process. Here we show that wild type PS1 stimulates the transcriptional activity ability of p300 whereas an Alzheimer's disease-associated mutant of PS1 did not produce this effect. To our knowledge, this is the first report that shows regulation of p300 activity by WT PS1 and not by mutant PS1, indicating that like CBP, p300 can be differentially regulated by WT PS1 compared to its AD-associated mutant.

The neonatal splice variant of Nav1.5 potentiates in vitro invasive behaviour of MDA-MB-231 human breast cancer cells.

Upregulation of functional voltage-gated Na+ channels (VGSCs) occurs in metastatic human breast cancer (BCa) in vitro and in vivo. The present study aimed to ascertain the specific involvement of the "neonatal" splice variant of Nav1.5 (nNav1.5), thought to be predominant, in the VGSC-dependent invasive behaviour of MDA-MB-231 cells. Functional activity of nNav1.5 was suppressed by two different methods targeting nNav1.5: (i) small interfering RNA (siRNA), and (ii) a polyclonal antibody (NESO-pAb); effects upon migration and invasion were determined. nNav1.5 mRNA, protein and signalling were measured using real-time PCR, Western blotting, and patch clamp recording, respectively. Treatment with the siRNA rapidly reduced (by approximately 90%) the level of nNav1.5 (but not adult Nav1.5) mRNA, but the protein reduction was much smaller (approximately 30%), even after 13 days. Nevertheless, the siRNA reduced peak VGSC current density by 33%, and significantly increased the cells' sensitivity to nanomolar tetrodotoxin (TTX). Importantly, the siRNA suppressed in vitro migration by 43%, and eliminated the normally inhibitory effect of TTX. Migrated MDA-MB-231 cells expressed more nNav1.5 protein at the plasma membrane than non-migrated cells. Furthermore, NESO-pAb reduced migration by up to 42%, in a dose-dependent manner. NESO-pAb also reduced Matrigel invasion without affecting proliferation. TTX had no effect on cells already treated with NESO-pAb. It was concluded that nNav1.5 is primarily responsible for the VGSC-dependent enhancement of invasive behaviour in MDA-MB-231 cells. Accordingly, targeting nNav1.5 expression/activity may be useful in clinical management of metastatic BCa.

Voltage-gated sodium channel expression and potentiation of human breast cancer metastasis.

PURPOSE: Ion channel activity is involved in several basic cellular behaviors that are integral to metastasis (e.g., proliferation, motility, secretion, and invasion), although their contribution to cancer progression has largely been ignored. The purpose of this study was to investigate voltage-gated Na(+) channel (VGSC) expression and its possible role in human breast cancer. EXPERIMENTAL DESIGN: Functional VGSC expression was investigated in human breast cancer cell lines by patch clamp recording. The contribution of VGSC activity to directional motility, endocytosis, and invasion was evaluated by in vitro assays. Subsequent identification of the VGSC alpha-subunit(s) expressed in vitro was achieved using reverse transcription-PCR, immunocytochemistry, and Western blot techniques and used to investigate VGSCalpha expression and its association with metastasis in vivo. RESULTS: VGSC expression was significantly up-regulated in metastatic human breast cancer cells and tissues, and VGSC activity potentiated cellular directional motility, endocytosis, and invasion. Reverse transcription-PCR revealed that Na(v)1.5, in its newly identified "neonatal" splice form, was specifically associated with strong metastatic potential in vitro and breast cancer progression in vivo. An antibody specific for this form confirmed up-regulation of neonatal Na(v)1.5 protein in breast cancer cells and tissues. Furthermore, a strong correlation was found between neonatal Na(v)1.5 expression and clinically assessed lymph node metastasis. CONCLUSIONS: Up-regulation of neonatal Na(v)1.5 occurs as an integral part of the metastatic process in human breast cancer and could serve both as a novel marker of the metastatic phenotype and a therapeutic target.

A novel polyclonal antibody specific for the Na(v)1.5 voltage-gated Na(+) channel 'neonatal' splice form.

Voltage-gated Na(+) channel (VGSC) diversity is achieved through a number of mechanisms: multiple subunits, multiple genes encoding the pore-forming VGSC alpha-subunit and multiple gene isoforms generated by alternative splicing. A major type of VGSCalpha alternative splicing is in D1:S3, which has been proposed to be developmentally regulated. We recently reported a D1:S3 spliced form of Na(v)1.5 in human metastatic breast cancer cells. This novel 'neonatal' isoform differs from the counterpart 'adult' form at seven amino acids (in the extracellular loop between S3-S4 of D1). Here, we generated an anti-peptide polyclonal antibody, named NESOpAb, which specifically recognised 'neonatal' but not 'adult' Na(v)1.5 when tested on cells specifically over-expressing one or other of these Na(v)1.5 spliced forms. The antibody was used to investigate developmental expression of 'neonatal' Na(v)1.5 (nNa(v)1.5) in a range of mouse tissues by immunohistochemistry. Overall, the results were consistent with nNa(v)1.5 protein being more abundantly expressed in selected tissues (particularly heart and brain) from neonate as compared to adult animals. Importantly, NESOpAb blocked functional nNa(v)1.5 ion conductance when applied extracellularly at concentrations as low as 0.05 ng/ml. Possible biological and clinical applications of NESOpAb are discussed.

T-lymphocyte invasiveness: control by voltage-gated Na+ channel activity.

Whole-cell patch-clamp recordings showed that a sub-population (10%) of Jurkat cells, a model of human T-cells, expressed a functional voltage-gated sodium channel, which was tetrodotoxin (TTX)-resistant. Expression of voltage-gated sodium channel protein was confirmed by western blots. Semi-quantitative PCR analysis revealed that mRNAs for the alpha-subunits of multiple voltage-gated sodium channel subtypes were present but indicated that Na(v)1.5 was the predominant subtype, consistent with the TTX-resistant nature of the recorded currents. Importantly, 10 microM TTX reduced the number of Jurkat cells invading a Matrigel basement membrane by 93.0+/-5.5%. Since similar sodium channels have also been detected in normal human T-lymphocytes, it is concluded that the activity of voltage-gated sodium channels could represent a novel mechanism potentiating the invasive capacity of these cells.