PYK2/PDZ-RhoGEF Links Ca(2+) Signaling to RhoA

Objective-Ras homolog gene family member A (RhoA)/Rho-kinase-mediated Ca(2+) sensitization is a critical component of constrictor responses. The present study investigates how angiotensin II activates RhoA. Methods and Results-Adenoviral vectors were used to manipulate the expression of regulator of G protein signaling (RGS) domain containing Rho-specific guanine exchange factors (RhoGEFs) and proline-rich tyrosine kinase 2 (PYK2), a nonreceptor tyrosine kinase, in primary rat vascular smooth muscle cells. As an evidence of RhoA activation, RhoA translocation and MYPT1 (the regulatory subunit of myosin light chain phosphatase) phosphorylation were analyzed by Western blot. Results showed that overexpression of PDZ-RhoGEF, but not p115-RhoGEF or leukemia-associated RhoGEF (LARG), enhanced RhoA activation by angiotensin II. Knockdown of PDZ-RhoGEF decreased RhoA activation by angiotensin II. PDZ-RhoGEF was phosphorylated and activated by PYK2 in vitro, and knockdown of PDZ-RhoGEF reduced RhoA activation by constitutively active PYK2, indicating that PDZ-RhoGEF links PYK2 to RhoA. Knockdown of PYK2 or PDZ-RhoGEF markedly decreased RhoA activation by A23187, a Ca(2+) ionophore, demonstrating that PYK2/PDZ-RhoGEF couples RhoA activation to Ca(2+). Conclusions-PYK2 and PDZ-RhoGEF are necessary for angiotensin II-induced RhoA activation and for Ca(2+) signaling to RhoA. (Arterioscler Thromb Vasc Biol. 2009;29:1657-1663.)

mRNA expression of matrix metalloproteinases (MMPs) 2 and 9 and tissue inhibitor of matrix metalloproteinases (TIMPs) 1 and 2 in childhood acute lymphoblastic leukemia: Potential role of TIMP1 as an adverse prognostic factor

This study evaluates the mRNA expression profile of genes TIMP1, TIMP2, MMP2 and MMP9 in diagnostic bone marrow samples from 134 consecutive ALL children by real-time quantitative PCR. A significant association was observed between higher expression levels of MMP9 and low risk group and absence of extramedullary infiltration and higher expression levels of TIMP2 and MMP2 with T-ALL. TIMP1 gene expression values higher than the median were associated with a significantly lower 5-year event free-survival in univariable (P = 0.04) and multivariable analysis (P = 0.01). Our data address new information in the complex interaction of the migration/adhesion genes and childhood ALL. (c) 2009 Elsevier Ltd. All rights reserved.

Chronic Ethanol Consumption Induces Cavernosal Smooth Muscle Dysfunction in Rats

OBJECTIVES To investigate the effects of chronic ethanol consumption on nitric oxide (NO)-mediated relaxation in rat cavernosal smooth muscle (CSM). METHODS Male wistar rats were divided into 2 groups: control and ethanol. CSM obtained from both groups were mounted in organ chambers for measurement of isometric tension. Contraction of the strips was induced by electrical field stimulation (EFS, 1-32 Hertz) and phenylephrine. We also evaluated the effect of ethanol consumption on the relaxation induced by acetylcholine (0.01-1000 mu mol L(-1)), sodium nitroprusside (SNP, 0.01-1000 mu mol L(-1)), or EFS (1-32 Hz) in strips precontracted with phenylephrine (10 mu mol L(-1)). Blood ethanol, serum testosterone levels, and basal nitrate generation were determined. Immunoexpression of endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) was also accessed. RESULTS Ethanol intake for 4 weeks significantly increased noradrenergic nerve-mediated contractions of CSM in response to EFS. The endothelium-dependent relaxation induced by acetylcholine decreased after the ethanol treatment. Ethanol consumption decreased serum testosterone levels but did not affect the nitrate levels on rat CSM. The mRNA and protein levels for eNOS and iNOS receptors were increased in CSM from ethanol-treated rats. CONCLUSIONS Ethanol consumption reduces endothelium-dependent relaxation induced by acetylcholine, but does not affect SNP or EFS-induced relaxation, suggesting that ethanol disrupts the endothelial function. Despite the overexpression of eNOS and iNOS in ethanol-treated rats, the impaired relaxation induced by acetylcholine may suggest that chronic ethanol consumption induces endothelial dysfunction. UROLOGY 74: 1250-1256, 2009. (C) 2009 Published by Elsevier Inc.

Precise Temporal Regulation of roughest is Required for Correct Salivary Gland Autophagic Cell Death in Drosophila

The Drosophila roughest (rst) locus encodes an immunoglobulin superfamily transmembrane glycoprotein implicated in a variety of embryonic and postembryonic developmental processes. Here we demonstrate a previously unnoticed role for this gene in the autophagic elimination of larval salivary glands during early pupal stages by showing that overexpression of the Rst protein ectodomain in early pupa leads to persistence of salivary glands up to at least 12 hours after head eversion, although with variable penetrance. The same phenotype is observed in individuals carrying the dominant regulatory allele rst(D), but not in loss of function alleles. Analysis of persistent glands at the ultrastructural level showed that programmed cell death starts at the right time but is arrested at an early stage of the process. Finally we describe the expression pattern and intracellular distribution of Rst in wild type and rstD mutants, showing that its downregulation in salivary glands at the beginning of pupal stage is an important factor in the correct implementation of the autophagic program of this tissue in space and time. genesis 47:492-504, 2009. (C) 2009 Wiley-Liss, Inc.

Chronic alcoholism associated with diabetes impairs erectile function in rats

OBJECTIVE To investigate the effects of chronic ethanol consumption and diabetes on nitric oxide (NO)-mediated relaxation of cavernosal smooth muscle (CSM). MATERIAL AND METHODS Male Wistar rats were divided into four groups: control, isocaloric, diabetic and ethanol-diabetic. The CSMs were mounted in organ chambers for measurement of isometric tension. Contraction of the strips was induced by electrical field stimulation (EFS, 1-32 Hz) and phenylephrine. We also evaluated the effect of ethanol consumption on the relaxation induced by acetylcholine (ACh; 0.01-1000 mu mol/L), sodium nitroprusside (SNP, 0.01-1000 mu mol/L) or EFS (1-32 Hz) in strips pre-contracted with phenylephrine (10 mu mol/L). Immunoexpression of endothelial NO synthase (eNOS) and inducible NOS (iNOS) was also accessed. RESULTS The endothelium-dependent relaxation induced by ACh was decreased in CSM from ethanol-diabetic rats when compared with the controls, with a mean (sem) of 21 (4) vs 37 (2)%. Similarly, the potency and maximal responses induced by SNP were reduced in the ethanol-diabetic [3.97 (0.38) and 85 (1)%, respectively] and diabetic groups [3.78 (0.56) and 81 (2)%, respectively] when compared with the controls [5.3 (0.22) and 90 (3)%, respectively] and isocaloric [5.3 (0.19) and 92 (1)%, respectively] groups. Noradrenergic nerve-mediated contractions of CSM in response to EFS were increased in rats from ethanol-diabetic and diabetic groups when compared with the control and isocaloric groups. Conversely, there were no differences in EFS-induced relaxation among the groups. The immunostaining assays showed overexpression of eNOS and iNOS in the CSM from diabetic and ethanol-diabetic rats when compared with the control and isocaloric rats. CONCLUSION There was an impairment of relaxation of CSM from ethanol-diabetic and diabetic rats that involved a decrease in the NO-cyclic guanosine monophosphate signalling pathway by endothelium-dependent mechanisms accompanied by a change in the CSM contractile sensitivity.

Fibrinolytic activity is associated with presence of cystic medial degeneration in aneurysms of the ascending aorta

Fibrinolytic activity is associated with presence of cystic medial degeneration in aneurysms of the ascending aorta Aims: Thoracic ascending aortic aneurysms (TAA) are characterized by elastic fibre breakdown and cystic medial degeneration within the aortic media, associated with progressive smooth muscle cell (SMC) rarefaction. The transforming growth factor (TGF)-beta/Smad2 signalling pathway is involved in this process. Because the pericellular fibrinolytic system activation is able to degrade adhesive proteins, activate matrix metalloproteinase (MMP), induce SMC disappearance and increase the bioavailability of TGF-beta, the aim was to investigate the plasminergic system in TAA. Methods and results: Ascending aortas [21 controls and 19 TAAs (of three different aetiologies)] were analysed. Immunohistochemistry showed accumulation of t-PA, u-PA and plasmin in TAAs, associated with residual SMCs. Overexpression of t-PA and u-PA was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting and zymography on TAA extracts and culture medium conditioned by TAA. Plasminogen was present on the SMC surface and inside cytoplasmic vesicles, but plasminogen mRNA was undetectable in the TAA medial layer. Plasmin-antiplasmin complexes were detected in TAA-conditioned medium and activation of the fibrinolytic system was associated with increased fibronectin turnover. Fibronectin-related material was detected immunohistochamically in dense clumps around SMCs and colocalized with latent TGF-beta binding protein-1. Conclusions: The fibrinolytic pathway could play a critical role in TAA progression, via direct or indirect impact on ECM and consecutive modulation of TGF-beta bioavailability.

Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan

Large, long-lived species experience more lifetime cell divisions and hence a greater risk of spontaneous tumor formation than smaller, short-lived species. Large, long-lived species are thus expected to evolve more elaborate tumor suppressor systems. In previous work, we showed that telomerase activity coevolves with body mass, but not lifespan, in rodents: telomerase activity is repressed in the somatic tissues of large rodent species but remains active in small ones. Without telomerase activity, the telomeres of replicating cells become progressively shorter until, at some critical length, cells stop dividing. Our findings therefore suggested that repression of telomerase activity mitigates the increased risk of cancer in larger-bodied species but not necessarily longer-lived ones. These findings imply that other tumor suppressor mechanisms must mitigate increased cancer risk in long-lived species. Here, we examined the proliferation of fibroblasts from 15 rodent species with diverse body sizes and lifespans. We show that, consistent with repressed telomerase activity, fibroblasts from large rodents undergo replicative senescence accompanied by telomere shortening and overexpression of p16(Ink4a) and p21(Cip1/Waf1) cycline-dependent kinase inhibitors. Interestingly, small rodents with different lifespans show a striking difference: cells from small shorter-lived species display continuous rapid proliferation, whereas cells from small long-lived species display continuous slow proliferation. We hypothesize that cells of small long-lived rodents, lacking replicative senescence, have evolved alternative tumor-suppressor mechanisms that prevent inappropriate cell division in vivo and slow cell growth in vitro. Thus, large-bodied species and small but long-lived species have evolved distinct tumor suppressor mechanisms.

Claudin expression is dysregulated in prostate adenocarcinomas but does not correlate with main clinicopathological parameters

Aims: Claudins, a large family of essential tight junction (TJ) proteins, are abnormally regulated in human carcinomas. The aim of this study was to determine the expression of claudins 1, 2, 3, 4, 5, 7, and 11 in prostate samples from Brazilian patients and correlate it with the clinicopathological features of prostate cancer. Methods: Using a tissue microarray (TMA) of specimens of prostate adenocarcinoma and benign prostatic hyperplasia (BPH) we analysed the expression of claudins 1, 2, 3, 4, 5, 7, and 11 by immunohistochemistry. Results: Claudin 4 was down-regulated and claudins 2, 3, and 5 were overexpressed in prostate adenocarcinomas compared with BPH samples. Expression of claudins 1 and 7 was similar in tumours and BPH samples. Claudin 11 was absent from all prostate samples. Overexpression of claudin 3 was associated with perineural invasion (p = 0.014) and tended to occur in advanced stages of the disease (p = 0.064). Increased expression of claudin 5 was marginally associated with perineural invasion (p = 0.060). Conclusions: Our results suggest that alterations in claudin expression occur in prostate cancer cells, although we have not found an association with the main clinicopathological parameters.

A role for COX2-derived PGE2 and PGE2-receptor subtypes in head and neck squamous carcinoma cell proliferation

The overexpression of cyclooxygenase (COX)-2 is a frequent event in squamous cell carcinomas of the head and neck (HNSCC), and non-steroidal anti-inflammatory drugs, which are potent inhibitors of COX-1 and COX-2, exert chemopreventive effects on HNSCC cancer development. COX-2 promotes the release of the pro-inflammatory mediator prostaglandin E2 (PGE2), which acts on its cell surface G protein-coupled receptors EP1, EP2, EP3, and EP4. Here, we investigated the role of PGE2 and its receptors in cellular proliferation in HNSCC. The expression of COX-2 and EP1-4 was examined in immortalized oral epithelial cells and in a representative panel of HNSCC cell lines, and based on these data EP1-EP3 and COX-2 expression were evaluated by immunohistochemistry in a large clinical sample collection using HNSCC tissue microarrays. The ability of selective COX-2 inhibition to block PGE2 secretion was measured by ELISA specific assays. The effects of PGE2 on cell proliferation were evaluated using PGE2, its stable analog, and EP2 and EP3-specific synthetic agonists. The results presented here show that HNSCC tumoral lesions and their derived cell lines constitutively express COX-2 and the EP1, EP2 and EP3 receptors for PGE2. HNSCC cells secrete PGE2, which can be suppressed by low concentrations of COX-2 selective inhibitors, without inhibiting cell proliferation. Exogenously added stable PGE2 and EP3-specific agonists induce DNA synthesis in all HNSCC cell lines tested. Overall, our study supports the emerging notion that PGE2 produced in the tumor microenvironment by the overexpression of COX-2 in tumoral and inflammatory cells may promote the growth of HNSCC cells in an autocrine and paracrine fashion by acting on PGE2 receptors that are widely expressed in most HNSCC cancer cells. In particular, our findings suggest that EP3 receptor may play a more prominent role in HNSCC cell growth promotion, thus providing a rationale for the future evaluation of this PGE2 receptor as a target for HNSCC prevention strategies. Published by Elsevier Ltd.

Differential expression of galectin-3, beta-catenin, and cyclin D1 in adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma of salivary glands

Background: Galectin-3 has been implicated in tumor progression of some malignancies as thyroid, prostate, and salivary gland tumors. Recently, it has been suggested that this protein may be an important mediator of the beta-catenin/Wnt pathway. Moreover, nuclear galectin-3 expression has been implicated in cell proliferation, promoting cyclin D1 activation. Thus, the present study aimed to correlate galectin-3 expression with beta-catenin and cyclin D1 expressions in adenoid cystic carcinoma (ACC) and in polymorphous low-grade adenocarcinoma (PLGA). Methods: Fifteen formalin-fixed paraffin-embedded cases of each tumor were retrieved from the files of the Surgical Oral Pathology Service at the University of Sao Paulo and the proteins were analyzed by immunohistochemistry. Results: Adenoid cystic carcinoma showed galectin-3 immunostaining mainly in the nuclei, while PLGA revealed a positive mostly cytoplasmic reaction to galectin-3 in the largest part of tumor cells. Both tumors showed intense cytoplasmic/nuclear staining for beta-catenin in majority of cases. Cyclin D1 immunoreactivity was not detected in 14/15 PLGA and showed specific nuclear staining in 10/15 cases of ACC in more than 5% of the neoplastic cells. Cyclin D1 expression was correlated with cytoplasmic and nuclear galectin-3 expression in ACC (P < 0.05). Conclusions: These results suggest that in ACC galectin-3 may play a role in cellular proliferation through cyclin D1 activation. In addition, nuclear expression of galectin-3 in ACC may be related to a more aggressive behavior of this lesion. Although beta-catenin seems to play a role in carcinogenesis in both lesions, it seems that it does not bind to galectin-3 for cyclin D1 stimulation.

Interrelationship between Epstein-Barr virus infection in gastric carcinomas and the expression of apoptosis-associated proteins

Aims: Epstein-Barr virus (EBV) and its associated proteins may be protective against the occurrence of apoptosis that would normally inhibit cancer development and progression. Alternatively, the viral infection may cause altered or mutated expression of oncogenes or tumour suppressor genes that are necessary for tumour development. an action that may also involve apoptosis, In this study, a relationship was sought between occurrence of EBV infection, expression of apoptosis-associated proteins (tumour suppressor gene p53 and oncogenes c-myc and bcl-2) and levels of cell death (apoptosis or necrosis) in 119 cases of gastric carcinoma. Methods and results: The EBV status of the gastric carcinomas (using the EBV-encoded small RNA I (EBER-1) and in-situ hybridization), stage and grade of tumour and sex of patients were compared for bcl-2, p53 and c-myc expression patterns. EBER-1 was detected in approximately 20% of cases studied. There was no significant correlation between levels of cell death in the tumour tissue and EBV status. In the protein analyses, development and progression of gastric carcinoma, with or without EBV infection. was independent of bcl-2 expression. However, in gastric cancers with EBV infection, p53 overexpression was inhibited and c-myc expression was increased in early stage cancers, in comparison with decreased c-myc expression in late stage cancers. Conclusions: The p53 and c-myc expression patterns indicate that EBV-infected gastric carcinomas are less likely to have a natural regression via apoptosis at an early stage and explain, in part, the resistance to treatment of late stage of gastric cancers.

Bril: A novel bone-specific modulator of mineralization

In the course of attempting to define the bone ""secretome"" using a signal-trap screening approach, we identified a gene encoding a small membrane protein novel to osteoblasts. Although previously identified in silico as ifitm5, no localization or functional studies had been undertaken on this gene. We characterized the expression patterns and localization of this gene in vitro and in vivo and assessed its role in matrix mineralization in vitro. The bone specificity and shown role in mineralization led us to rename the gene bone restricted ifitm-like protein (Bril). Bril encodes a 14.8-kDa 1.34 arnino acid protein with two transmembrane domains. Northern blot analysis showed bone-specific expression with no expression in other embryonic or adult tissues. In situ hybridization and immunohistochemistry in mouse embryos showed expression localized on the developing bone. Screening of cell lines showed Bril expression to be highest in osteoblasts, associated with the onset of matrix maturation/mineralization, suggesting a role in bone formation. Functional evidence of a role in mineralization was shown by adenovirus-mediated Brit overexpression and lentivirus-mediated Bril shRNA knockdown in vitro. Elevated Bril resulted in dose-dependent increases in mineralization in UMR106 and rat primary osteoblasts. Conversely, knockdown of Bril in MC3T3 osteoblasts resulted in reduced mineralization. Thus, we identified Bril as a novel osteoblast protein and showed a role in mineralization, possibly identifying a new regulatory pathway in bone formation.

Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts

Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts, This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones, This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types, This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.

Murine ventricular L-type Ca2+ current is enhanced by zinterol via beta(1)-adrenoceptors, and is reduced in TG4 mice overexpressing the human beta(2)-adrenoceptor

1 The functional coupling of B-2-adrenoceptors (beta (2)-ARs) to murine L-type Ca2+ current (I-Ca(L)) was investigated with two different approaches. The beta (2)-AR signalling cascade was activated either with the beta (2)-AR selective agonist zinterol (myocytes from wild-type mice), or by spontaneously active, unoccupied beta (2)-ARs (myocytes from TG4 mice with 435 fold overexpression of human beta (2)-ARs). Ca2+ and Ba2+ currents were recorded in the whole-cell and cell-attached configuration of the patch- clamp technique, respectively. 2 Zinterol (10 muM) significantly increased I-Ca(L) amplitude of wild-type myocytes by 19+/-5%, and this effect was markedly enhanced after inactivation of Gi-proteins with pertussis-toxin (PTX; 76+/-13% increase). However, the effect of zinterol was entirely mediated by the beta (1)-AR subtype, since it was blocked by the beta (1)-AR selective antagonist CGP 20712A (300 nM). The beta (2)-AR selective antagonist ICI 118,551 (50 nM) did not affect the response of I-Ca(L) to zinterol. 3 In myocytes with beta (2)-AR overexpression I-Ca(L) was not stimulated by the activated signalling cascade. On the contrary, I-Ca(L) was lower in TG4 myocytes and a significant reduction of single-channel activity was identified as a reason for the lower whole-cell I-Ca(L). The beta (2)-AR inverse agonist ICI 118,551 did not further decrease I-Ca(L). PTX-treatment increased current amplitude to values found in control myocytes. 4 In conclusion, there is no evidence for beta (2)-AR mediated increases of I-Ca(L) in wild-type mouse ventricular myocytes. Inactivation of Gi-proteins does not unmask beta (2)-AR responses to zinterol, but augments beta (1)-AR mediated increases of I-Ca(L). In the mouse model of beta (2)-AR overexpression I-Ca(L) is reduced due to tonic activation of Gi-proteins.

Transactivation-deficient p73 alpha (p73 Delta exon2) inhibits apoptosis and competes with p53

p73 has recently been identified as a structural and functional homolog of the tumor suppressor protein p53. Overexpression of p53 activates transcription of p53 effector genes, causes growth inhibition and induced apoptosis. We describe here the effects of a tumor-derived truncated transcript of p73 alpha (p73 Delta exon2) on p53 function and on cell death. This transcript, which lacks the acidic N-terminus corresponding to the transactivation domain of p53, was initially detected in a neuroblastoma cell line. Overexpression of p73 Delta exon2 partially protects lymphoblastoid cells against apoptosis induced by anti-Fas antibody or cisplatin. By cotransfecting p73 Delta exon2 with wild-type p53 in the p53 null line Saos 2, we found that this truncated transcript reduces the ability of wild-type p53 to promote apoptosis. This anti-apoptotic effect was also observed when p73 Delta exon2 was co-transfected with full-length p73 (p73 alpha). This was further substantiated by suppression of p53 transactivation of the effector gene p21-Waf1 in p73 Delta exon2 transfected cells and by inhibition of expression of a reporter gene under the control of the p53 promoter. Thus, this truncated form of p73 can act as a dominant-negative agent towards transactivation by p53 and p73 alpha, highlighting the potential implications of these findings for p53 signaling pathway. Furthermore, we demonstrate the existence of a p73 Delta exon2 transcript in a very significant proportion (46%) of breast cancer cell lines. However, a large spectrum of normal and malignant tissues need to be surveyed to determine whether this transdominant p73 variant occurs in a tumor-specific manner.