Cristina G. Viloria

Modulation of human erg K+ channel gating by activation of a G protein‐coupled receptor and protein kinase C

1 Modulation of the human ether‐à‐go‐go‐related gene (HERG) K+ channel was studied in two‐electrode voltage‐clamped Xenopus oocytes co‐expressing the channel protein and the thyrotropin‐releasing hormone (TRH) receptor. 2 Addition of TRH caused clear modifications of HERG channel gating kinetics. These variations consisted of an acceleration of deactivation, as shown by a faster decay of hyperpolarization‐induced tail currents, and a slower time course of activation, measured using an envelope of tails protocol. The voltage dependence for activation was also shifted by nearly 20 mV in the depolarizing direction. Neither the inactivation nor the inactivation recovery rates were altered by TRH. 3 The alterations in activation gating parameters induced by TRH were demonstrated in a direct way by looking at the increased outward K+ currents elicited in extracellular solutions in which K+ was replaced by Cs+. 4 The effects of TRH were mimicked by direct pharmacological activation of protein kinase C (PKC) with β‐phorbol 12‐myristate, 13‐acetate (PMA). The TRH‐induced effects were antagonized by GF109203X, a highly specific inhibitor of PKC that also abolished the PMA‐dependent regulation of the channels. 5 It is concluded that a PKC‐dependent pathway links G protein‐coupled receptors that activate phospholipase C to modulation of HERG channel gating. This provides a mechanism for the physiological regulation of cardiac function by phospholipase C‐activating receptors, and for modulation of adenohypophysial neurosecretion in response to TRH.

Differential effects of amino-terminal distal and proximal domains in the regulation of human erg K(+) channel gating.

The participation of amino-terminal domains in human ether-a-go-go (eag)-related gene (HERG) K(+) channel gating was studied using deleted channel variants expressed in Xenopus oocytes. Selective deletion of the HERG-specific sequence (HERG Delta138-373) located between the conserved initial amino terminus (the eag or PAS domain) and the first transmembrane helix accelerates channel activation and shifts its voltage dependence to hyperpolarized values. However, deactivation time constants from fully activated states and channel inactivation remain almost unaltered after the deletion. The deletion effects are equally manifested in channel variants lacking inactivation. The characteristics of constructs lacking only about half of the HERG-specific domain (Delta223-373) or a short stretch of 19 residues (Delta355-373) suggest that the role of this domain is not related exclusively to its length, but also to the presence of specific sequences near the channel core. Deletion-induced effects are partially reversed by the additional elimination of the eag domain. Thus the particular combination of HERG-specific and eag domains determines two important HERG features: the slow activation essential for neuronal spike-frequency adaptation and maintenance of the cardiac action potential plateau, and the slow deactivation contributing to HERG inward rectification.

Genetic inactivation of ADAMTS15 metalloprotease in human colorectal cancer.

Matrix metalloproteinases have been traditionally linked to cancer dissemination through their ability to degrade most extracellular matrix components, thus facilitating invasion and metastasis of tumor cells. However, recent functional studies have revealed that some metalloproteases, including several members of the ADAMTS family, also exhibit tumor suppressor properties. In particular, ADAMTS1, ADAMTS9, and ADAMTS18 have been found to be epigenetically silenced in malignant tumors of different sources, suggesting that they may function as tumor suppressor genes. Herein, we show that ADAMTS15 is genetically inactivated in colon cancer. We have performed a mutational analysis of the ADAMTS15 gene in human colorectal carcinomas, with the finding of four mutations in 50 primary tumors and 6 colorectal cancer cell lines. Moreover, functional in vitro and in vivo studies using HCT-116 and SW-620 colorectal cancer cells and severe combined immunodeficient mice have revealed that ADAMTS15 restrains tumor growth and invasion. Furthermore, the presence of ADAMTS15 in human colorectal cancer samples showed a negative correlation with the histopathologic differentiation grade of the corresponding tumors. Collectively, these results provide evidence that extracellular proteases, including ADAMTS15, may be targets of inactivating mutations in human cancer and further validate the concept that secreted metalloproteases may show tumor suppressor properties.

The ADAMTS12 metalloprotease gene is epigenetically silenced in tumor cells and transcriptionally activated in the stroma during progression of colon cancer

Proteases have long been associated with tumor progression, given their ability to degrade extracellular matrix components and facilitate invasion and metastasis. However, recent findings indicate that different proteases can also act as tumor-suppressor enzymes. We have recently reported that lung carcinoma cells expressing the ADAMTS-12 metalloprotease show a remarkable impairment of growth in immunodeficient mice as compared with parental cells. Here, we show that ADAMTS12 promoter is hypermethylated in cancer cell lines and tumor tissues. Interestingly, ADAMTS12 expression in the stromal cells surrounding epithelial malignant cells is higher than in the paired normal tissues. Moreover, the expression of this metalloprotease in colon fibroblasts co-cultured with colon cancer cell lines is higher than in those cultured alone. Furthermore, the expression of ADAMTS-12 by these fibroblasts is linked with an anti-proliferative effect on tumor cells. Based on these findings, we hypothesize that ADAMTS-12 is a novel anti-tumor protease that can reduce the proliferative properties of tumor cells. This function is lost by epigenetic silencing in tumor cells, but concurrently induced in stromal cells, probably as part of a response of the normal tissue aimed at controlling the progression of cancer.

Mutational and Functional Analysis Reveals ADAMTS18 Metalloproteinase as a Novel Driver in Melanoma

The disintegrin-metalloproteinases with thrombospondin domains (ADAMTS) genes have been suggested to function as tumor suppressors as several have been found to be epigenetically silenced in various cancers. We performed a mutational analysis of the ADAMTS gene family in human melanoma and identified a large fraction of melanomas to harbor somatic mutations. To evaluate the functional consequences of the most commonly mutated gene, ADAMTS18, six of its mutations were biologically examined. ADAMTS18 mutations had little effect on melanoma cell growth under standard conditions, but reduced cell dependence on growth factors. ADAMTS18 mutations also reduced adhesion to laminin and increased migration in vitro and metastasis in vivo. Melanoma cells expressing mutant ADAMTS18 had reduced cell migration after short hairpin RNA–mediated knockdown of ADAMTS18, suggesting that ADAMTS18 mutations promote growth, migration, and metastasis in melanoma. Mol Cancer Res; 8(11); 1513–25. ©2010 AACR.

Relevance of the proximal domain in the amino‐terminus of HERG channels for regulation by a phospholipase C‐coupled hormone receptor

We used Xenopus oocytes co‐expressing thyrotropin‐releasing hormone (TRH) receptors and human ether‐a‐go‐go‐related gene (HERG) K+ channel variants carrying different amino‐terminal modifications to check the relevance of the proximal domain for hormonal regulation of the channel. Deletion of the whole proximal domain (Δ138–373) eliminates TRH‐induced modifications in activation and deactivation parameters. TRH effects on activation are also suppressed with channels lacking the second half of the proximal domain or only residues 326–373. However, normal responses to TRH are obtained with Δ346–373 channels. Thus, whereas residues 326–345 are required for the hormonal modulation of HERG activation, different proximal domain sequences contribute to set HERG gating characteristics and its regulation by TRH.

Role of Periostin in Adhesion and Migration of Bone Remodeling Cells

Periostin is an extracellular matrix protein highly expressed in collagen-rich tissues subjected to continuous mechanical stress. Functionally, periostin is involved in tissue remodeling and its altered function is associated to numerous pathological processes. In orthodontics, periostin plays key roles in the maintenance of dental tissues and it is mainly expressed in those areas where tension or pressing forces are taking place. In this regard, high expression of periostin is essential to promote migration and proliferation of periodontal ligament fibroblasts. However little is known about the participation of periostin in migration and adhesion processes of bone remodeling cells. In this work we employ the mouse pre-osteoblastic MC3T3-E1 and the macrophage-like RAW 264.7 cell lines to overexpress periostin and perform different cell-based assays to study changes in cell behavior. Our data indicate that periostin overexpression not only increases adhesion capacity of MC3T3-E1 cells to different matrix proteins but also hampers their migratory capacity. Changes on RNA expression profile of MC3T3-E1 cells upon periostin overexpression have been also analyzed, highlighting the alteration of genes implicated in processes such as cell migration, adhesion or bone metabolism but not in bone differentiation. Overall, our work provides new evidence on the impact of periostin in osteoblasts physiology.

The nutraceutical flavonoid luteolin inhibits ADAMTS-4 and ADAMTS-5 aggrecanase activities

A disintegrin and metalloprotease with thrombospondin domains (ADAMTS)-4 (aggrecanase-1) and ADAMTS-5 (aggrecanase-2) are metalloproteases involved in articular cartilage degradation and represent potential therapeutic targets in arthritis treatment. We explore herein the ability of different natural compounds to specifically block the destructive action of these enzymes. Following a preliminary screening using carboxymethylated transferrin as substrate, we focused our interest on luteolin due to its inhibitory effect on ADAMTS-4 and ADAMTS-5 activities using aggrecan and fluorogenic peptides as substrates. However, matrix metalloproteinases (MMPs) activities on these substrates result less affected by this flavonoid. Moreover, incubation of mouse chondrogenic ATDC5 cells in the presence of luteolin clearly decreases the release of aggrecan fragments mediated by aggrecanases under the same conditions in which aggrecanolysis mediated by MMPs is detected. Additionally, glycosaminoglycan levels in culture medium of murine cartilage explants stimulated with interleukin-1-alpha plus retinoic acid are reduced by the presence of the flavonoid. This inhibition takes place through blockade of ADAMTS-mediated aggrecanolysis, while MMPs activity is not or poorly affected. These results suggest that luteolin could be employed as a prototypic modifying disease-agent to create new chondroprotective compounds aimed to specifically block the unwanted aggrecanase activities in arthritic diseases.

Abstract 4970: Mutational and functional analysis reveals ADAMTS18 metalloproteinase as a novel driver in melanoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL An important feature of cellular signaling and migration involves the proteolytic cleavage of extracellular matrix and/or pro-ligands involved in signal transduction. Proteins called proteases are responsible for this action and include the matrix-metalloproteinase family and the A Disintegrin and Metalloproteinases family to name a few. The A Disintegrin and Metalloproteinases with Thrombospondin domains (ADAMTS) have been suggested to function as tumor suppressors as several have been found to be epigenetically silenced in various cancers. Thus further analysis is warranted in determining if ADAMTS genes/proteins play a role in melanoma genesis. In the present study we performed a mutational analysis of 19 ADAMTS gene family members in human melanoma and identified a large fraction of melanomas to harbor somatic mutations. To evaluate the functional consequences of the most commonly mutated gene, ADAMTS18, six of its mutations were biologically examined. ADAMTS18 mutations had little effect on melanoma cell growth under standard conditions, but reduced cell dependence on growth factors. ADAMTS18 mutations also reduced adhesion to Laminin-I and increased migration in vitro and metastasis in vivo. Melanoma cells expressing mutant ADAMTS18 had reduced cell migration after shRNA-mediated knockdown of ADAMTS18, suggesting that ADAMTS18 mutations are growth-, migration- and metastasis- promoting in melanoma. These results demonstrate that the ADAMTS18 is a major target for mutational activation in melanoma and may be a useful diagnostic and/or therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4970. doi:10.1158/1538-7445.AM2011-4970