Overexpression of cortactin in head and neck squamous cell carcinomas can be uncoupled from augmented EGF receptor expression.

BACKGROUND: The gene encoding cortactin, CTTN (locus 11q13), an actin-binding substrate of Src kinases, is frequently amplified in breast and head and neck squamous cell carcinomas (HNSCC) and cortactin overexpression is thought to contribute in a significant way to the invasive phenotype of these tumors. Elevated Epidermal Growth Factor receptor (EGFR) expression is also commonly observed in HNSCC and has been associated with poor prognosis and resistance to cytotoxic agents, including ionizing radiation. It has been suggested that cortactin overexpression may increase EGFR levels in these tumors by affecting receptor downregulation, however we recently found by multivariate analysis, that cortactin expression status remained an independent prognostic factor for local recurrence, disease-free survival, and overall survival. MATERIAL AND METHODS: To examine the potential link between cortactin overexpression and EGFR status, we compared cortactin and EGFR levels in a series of tumor lines derived from HNSCC. RNAi-mediated silencing was performed in cortactin overexpressing cells and in vivo tumoral potential with respect to cortactin and EGFR status was analyzed. RESULTS AND DISCUSSION: Cortactin and EGFR levels were not strictly coupled in these lines and cortactin depletion did not decrease steady state receptor levels, although it did affect the epithelial to mesenchymal phenotypic conversion of cells. These results, together with clinical findings point to the existence of an EGFR-independent role of cortactin in HNSCC that may have important implications regarding the design of targeted therapies to combat tumor spread.

Functional ion channels in human pulmonary artery smooth muscle cells: Voltage-dependent cation channels.

The activity of voltage-gated ion channels is critical for the maintenance of cellular membrane potential and generation of action potentials. In turn, membrane potential regulates cellular ion homeostasis, triggering the opening and closing of ion channels in the plasma membrane and, thus, enabling ion transport across the membrane. Such transmembrane ion fluxes are important for excitation-contraction coupling in pulmonary artery smooth muscle cells (PASMC). Families of voltage-dependent cation channels known to be present in PASMC include voltage-gated K(+) (Kv) channels, voltage-dependent Ca(2+)-activated K(+) (Kca) channels, L- and T- type voltage-dependent Ca(2+) channels, voltage-gated Na(+) channels and voltage-gated proton channels. When cells are dialyzed with Ca(2+)-free K(+)- solutions, depolarization elicits four components of 4-aminopyridine (4-AP)-sensitive Kvcurrents based on the kinetics of current activation and inactivation. In cell-attached membrane patches, depolarization elicits a wide range of single-channel K(+) currents, with conductances ranging between 6 and 290 pS. Macroscopic 4-AP-sensitive Kv currents and iberiotoxin-sensitive Kca currents are also observed. Transcripts of (a) two Na(+) channel α-subunit genes (SCN5A and SCN6A), (b) six Ca(2+) channel α-subunit genes (α(1A), α(1B), α(1x), α(1D), α(1E) and α(1G)) and many regulatory subunits (α(2)δ(1), ß(1-4), and γ(6)), (c) 22 Kv channel α-subunit genes (Kv1.1 - Kv1.7, Kv1.10, Kv2.1, Kv3.1, Kv3.3, Kv3.4, Kv4.1, Kv4.2, Kv5.1, Kv 6.1-Kv6.3, Kv9.1, Kv9.3, Kv10.1 and Kv11.1) and three Kv channel ß-subunit genes (Kv(ß1-3) and (d) four Kca channel α-subunit genes (Sloα1 and SK2-SK4) and four Kca channel (ß-subunit genes (Kca(ß1-4) have been detected in PASMC. Tetrodotoxin-sensitive and rapidly inactivating Na(+) currents have been recorded with properties similar to those in cardiac myocytes. In the presence of 20 mM external Ca(2+), membrane depolarization from a holding potential of -100 mV elicits a rapidly inactivating T-type Ca(2+) current, while depolarization from a holding potential of -70 mV elicits a slowly inactivating dihydropyridine-sensitive L-type Ca(2+) current. This review will focus on describing the electrophysiological properties and molecular identities of these voltage-dependent cation channels in PASMC and their contribution to the regulation of pulmonary vascular function and its potential role in the pathogenesis of pulmonary vascular disease.

A new regulatory element mediates ethanol repression of KlADH3, a Kluyveromyces lactis gene coding for a mitochondrial alcohol dehydrogenase.

KlADH3 is a Kluyveromyces lactis alcohol dehydrogenase gene induced in the presence of all respiratory carbon sources except ethanol, which specifically represses this gene. Deletion analysis of the KlADH3 promoter revealed the presence of both positive and negative elements. However, by site-directed mutagenesis and gel retardation experiments, we identified a 15-bp element responsible for the transcriptional repression of this gene by ethanol. In particular, this element showed putative sites required for the sequential binding of ethanol-induced factors responsible for the repressed conditions, and the binding of additional factors relieved repression. In addition, we showed that the ethanol element was required for in vivo repression of KlAdh3 activity.

Bone morphogenetic protein-2 upregulates expression and function of voltage-gated K+ channels in human pulmonary artery smooth muscle cells.

Activity of voltage-gated K(+) (K(V)) channels in pulmonary artery smooth muscle cells (PASMC) plays an important role in control of apoptosis and proliferation in addition to regulating membrane potential and pulmonary vascular tone. Bone morphogenetic proteins (BMPs) inhibit proliferation and induce apoptosis in normal human PASMC, whereas dysfunctional BMP signaling and downregulated K(V) channels are involved in pulmonary vascular medial hypertrophy associated with pulmonary hypertension. This study evaluated the effect of BMP-2 on K(V) channel function and expression in normal human PASMC. BMP-2 (100 nM for 18-24 h) significantly (>2-fold) upregulated mRNA expression of KCNA5, KCNA7, KCNA10, KCNC3, KCNC4, KCNF1, KCNG3, KCNS1, and KCNS3 but downregulated (at least 2-fold) KCNAB1, KCNA2, KCNG2, and KCNV2. The most dramatic change was the >10-fold downregulation of KCNG2 and KCNV2, two electrically silent gamma-subunits that form heterotetramers with functional K(V) channel alpha-subunits (e.g., KCNB1-2). Furthermore, the amplitude and current density of whole cell K(V) currents were significantly increased in PASMC treated with BMP-2. It has been demonstrated that K(+) currents generated by KCNB1 and KCNG1 (or KCNG2) or KCNB1 and KCNV2 heterotetramers are smaller than those generated by KCNB1 homotetramers, indicating that KCNG2 and KCNV2 (2 subunits that were markedly downregulated by BMP-2) are inhibitors of functional K(V) channels. These results suggest that BMP-2 divergently regulates mRNA expression of various K(V) channel alpha-, beta-, and gamma-subunits and significantly increases whole cell K(V) currents in human PASMC. Finally, we present evidence that attenuation of c-Myc expression by BMP-2 may be involved in BMP-2-mediated increase in K(V) channel activity and regulation of K(V) channel expression. The increased K(V) channel activity may be involved in the proapoptotic and/or antiproliferative effects of BMP-2 on PASMC.

Identification of functional voltage-gated Na(+) channels in cultured human pulmonary artery smooth muscle cells.

Electrical excitability, which plays an important role in excitation-contraction coupling in the pulmonary vasculature, is regulated by transmembrane ion flux in pulmonary artery smooth muscle cells (PASMC). This study aimed to characterize the electrophysiological properties and molecular identities of voltage-gated Na(+) channels in cultured human PASMC. We recorded tetrodotoxin (TTX) sensitive and rapidly inactivating Na(+) currents with properties similar to those described in cardiac myocytes. Using RT-PCR, we detected transcripts of seven Na(+) channel alpha genes (SCN2A, 3A, 4A, 7A, 8A, 9A, and 11A), and two beta subunit genes (SCN1B and 2B). Our results demonstrate that human PASMC express TTX-sensitive voltage-gated Na(+) channels. Their physiological functions remain unresolved, although our data suggest that Na(+) channel activity does not directly influence membrane potential, intracellular Ca(2+) release, or proliferation in normal human PASMC. Whether their expression and/or activity are heightened in the pathological state is discussed.

Divergent effects of BMP-2 on gene expression in pulmonary artery smooth muscle cells from normal subjects and patients with idiopathic pulmonary arterial hypertension.

Bone morphogenetic proteins (BMPs) inhibit proliferation and induce apoptosis in pulmonary artery smooth muscle cells (PASMCs) from normal subjects. Dysfunction of BMP signaling due to mutations in and/or down-regulation of BMP receptors has been implicated in idiopathic pulmonary arterial hypertension (IPAH). The authors examined whether BMP differentially regulates gene expression in PASMCs from normal subjects and IPAH patients using the Affymetrix microarray analysis. BMP-2 treatment (200 nM for 24 hours) altered expression levels of 6206 genes in normal and IPAH PASMCs. Of these genes, 1063 were regulated oppositely by BMP-2: 523 genes were down-regulated by BMP-2 in normal PASMCs but up-regulated in IPAH PASMCs, whereas 540 genes were up-regulated by BMP-2 in normal PASMCs but down-regulated in IPAH PASMCs. The divergent effects of BMP-2 on gene expression profiles indicate that PASMCs may undergo significant phenotypic changes in IPAH patients during development of the disease. The transition of the antiproliferative effect of BMP-2 in normal PASMCs to its proliferative effect in IPAH patients is attributed potentially to its differential effect on expression patterns of various genes that are involved in cell proliferation and apoptosis. Among the 6206 BMP-2-sensitive genes, there are more than 1800 genes whose expression levels were negatively (correlation coefficient, r, <-0.9) or positively (with r >+ 0.9) correlated with the pulmonary arterial pressure. These results suggest that BMP-mediated gene regulation is significantly altered in PASMCs from IPAH patients and mRNA expression changes in BMP-regulated genes may be involved in the development of IPAH.

ATP-induced mitogenesis is mediated by cyclic AMP response element-binding protein-enhanced TRPC4 expression and activity in human pulmonary artery smooth muscle cells.

Extracellular ATP and intracellular cyclic AMP response element-binding protein (CREB, a transcription factor) promote cell proliferation in many cell types. The canonical transient receptor potential (TRPC) channels, which putatively participate in forming store- and receptor-operated Ca2+ channels, have been implicated in the pulmonary vascular remodeling processes. A link between extracellular ATP, CREB activation, and TRPC4 channel expression and activity has not been shown in human pulmonary artery smooth muscle cells (PASMC). Long-term (24-48 h) treatment of human PASMC with a low dose (100 microM) of ATP, which did not trigger a transient rise in free cytosolic Ca2+ concentration ([Ca2+]i) when applied acutely to the cells, caused marked increases in CREB phosphorylation and TRPC4 protein expression. The time course indicated that the ATP-mediated CREB phosphorylation preceded TRPC4 upregulation, whereas transfection of a nonphosphorylatable CREB mutant abolished ATP-mediated TRPC4 expression. Furthermore, treatment of human PASMC with ATP also enhanced the amplitude of capacitative Ca2+ entry (CCE) induced by passive store depletion, whereas the small interfering RNA specifically targeting TRPC4 attenuated ATP-mediated increases in TRPC4 expression and CCE amplitude and inhibited ATP-induced PASMC proliferation. These data suggest that low-dose ATP exerts part of its mitogenic effect in human PASMC via CREB-mediated upregulation of TRPC4 channel expression and activity and the subsequent increase in CCE and [Ca2+]i.

Hypoxia increases AP-1 binding activity by enhancing capacitative Ca2+ entry in human pulmonary artery endothelial cells.

Activating protein (AP)-1 transcription factors modulate expression of genes involved in cell proliferation and migration. Chronic hypoxia increases pulmonary artery smooth muscle cell proliferation by upregulating AP-1-responsive genes encoding for endothelium-derived vasoactive and mitogenic factors implicated in pulmonary hypertension development. The expression of AP-1 transcription factors is sensitive to changes in cytosolic free [Ca2+] ([Ca2+]cyt). Capacitative Ca2+ entry (CCE) via store-operated Ca2+ channels (SOC) is an important mechanism for raising [Ca2+]cyt in pulmonary artery endothelial cells (PAEC). Using combined molecular biological, fluorescence microscopy, and biophysical approaches, we examined the effect of chronic hypoxia (3% O2, 72 h) on AP-1 DNA binding activity, CCE, and transient receptor potential (TRP) gene expression in human (h) PAEC. EMSA showed that AP-1 binding to hPAEC nuclear protein extracts was significantly enhanced by hypoxia, the increase being dependent on store-operated Ca2+ influx and sensitive to La3+, an SOC inhibitor. Hypoxia also increased basal [Ca2+]cyt, the amount of CCE produced by store depletion with cyclopiazonic acid, and the amplitude of SOC-mediated currents (ISOC). The increases of CCE amplitude and ISOC current density by hypoxia were paralleled by enhanced TRPC4 mRNA and protein expression. Hypoxia-enhanced CCE and TRPC4 expression were also attenuated by La3+. These data suggest that hypoxia increases AP-1 binding activity by enhancing Ca2+ influx via La3+-sensitive TRP-encoded SOC channels in hPAEC. The Ca2+-mediated increase in AP-1 binding may play an important role in upregulating AP-1-responsive gene expression, in stimulating pulmonary vascular cell proliferation and, ultimately, in pulmonary vascular remodeling in patients with hypoxia-mediated pulmonary hypertension.

Diversity of voltage-dependent K+ channels in human pulmonary artery smooth muscle cells.

Electrical excitability, which plays an important role in excitation-contraction coupling in the pulmonary vasculature, is regulated by transmembrane ion flux in pulmonary artery smooth muscle cells (PASMC). This study examined the heterogeneous nature of native voltage-dependent K(+) channels in human PASMC. Both voltage-gated K(+) (K(V)) currents and Ca(2+)-activated K(+) (K(Ca)) currents were observed and characterized. In cell-attached patches of PASMC bathed in Ca(2+)-containing solutions, depolarization elicited a wide range of K(+) unitary conductances (6-290 pS). When cells were dialyzed with Ca(2+)-free and K(+)-containing solutions, depolarization elicited four components of K(V) currents in PASMC based on the kinetics of current activation and inactivation. Using RT-PCR, we detected transcripts of 1) 22 K(V) channel alpha-subunits (K(V)1.1-1.7, K(V)1.10, K(V)2.1, K(V)3.1, K(V)3.3-3.4, K(V)4.1-4.2, K(V)5.1, K(V) 6.1-6.3, K(V)9.1, K(V)9.3, K(V)10.1, and K(V)11.1), 2) three K(V) channel beta-subunits (K(V)beta 1-3), 3) four K(Ca) channel alpha-subunits (Slo-alpha 1 and SK2-SK4), and 4) four K(Ca) channel beta-subunits (K(Ca)beta 1-4). Our results show that human PASMC exhibit a variety of voltage-dependent K(+) currents with variable kinetics and conductances, which may result from various unique combinations of alpha- and beta-subunits forming the native channels. Functional expression of these channels plays a critical role in the regulation of membrane potential, cytoplasmic Ca(2+), and pulmonary vasomotor tone.

Bone morphogenetic proteins induce apoptosis in human pulmonary vascular smooth muscle cells.

Pulmonary vascular medial hypertrophy in primary pulmonary hypertension (PPH) is mainly caused by increased proliferation and decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs). Mutations of the bone morphogenetic protein (BMP) receptor type II (BMP-RII) gene have been implicated in patients with familial and sporadic PPH. The objective of this study was to elucidate the apoptotic effects of BMPs on normal human PASMCs and to examine whether BMP-induced effects are altered in PASMCs from PPH patients. Using RT-PCR, we detected six isoforms of BMPs (BMP-1 through -6) and three subunits of BMP receptors (BMP-RIa, -RIb, and -RII) in PASMCs. Treatment of normal PASMCs with BMP-2 or -7 (100-200 nM, 24-48 h) markedly increased the percentage of cells undergoing apoptosis. The BMP-2-mediated apoptosis in normal PASMCs was associated with a transient activation or phosphorylation of Smad1 and a marked downregulation of the antiapoptotic protein Bcl-2. In PASMCs from PPH patients, the BMP-2- or BMP-7-induced apoptosis was significantly inhibited compared with PASMCs from patients with secondary pulmonary hypertension. These results suggest that the antiproliferative effect of BMPs is partially due to induction of PASMC apoptosis, which serves as a critical mechanism to maintain normal cell number in the pulmonary vasculature. Inhibition of BMP-induced PASMC apoptosis in PPH patients may play an important role in the development of pulmonary vascular medial hypertrophy in these patients.

Enhanced expression of transient receptor potential channels in idiopathic pulmonary arterial hypertension.

Pulmonary vascular medial hypertrophy caused by excessive pulmonary artery smooth muscle cell (PASMC) proliferation is a major cause for the elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Increased Ca(2+) influx is an important stimulus for PASMC proliferation. Transient receptor potential (TRP) channel genes encode Ca(2+) channels that are responsible for Ca(2+) entry during cell proliferation. Normal human PASMC expressed multiple canonical TRP (TRPC) isoforms; TRPC6 was highly expressed and TRPC3 was minimally expressed. The protein expression of TRPC6 in normal PASMC closely correlated with the expression of Ki67, suggesting that TRPC6 expression is involved in the transition of PASMC from quiescent phase to mitosis. In lung tissues and PASMC from IPAH patients, the mRNA and protein expression of TRPC3 and -6 were much higher than in those from normotensive or secondary pulmonary hypertension patients. Inhibition of TRPC6 expression with TRPC6 small interfering RNA markedly attenuated IPAH-PASMC proliferation. These results demonstrate that expression of TRPC channels correlates with the progression of the cell cycle in PASMC. TRPC channel overexpression may be partially responsible for the increased PASMC proliferation and pulmonary vascular medial hypertrophy in IPAH patients.