Plant Mol Biol. 2009 Jul 24;
Li F, Shi J, Shen C, Chen G, Hu S, Chen Y

Elsholtzia splendens is generally considered as a Cu-tolerant and -accumulating plant species, and a candidate for phytoremediation of Cu-contaminated soils. To better understand the Cu tolerance/accumulation mechanisms in E. splendens, proteomic analysis was performed on E. splendens roots and leaves exposed to 100 muM CuSO(4) for 3 and 6 days. After 6 days of treatment, Cu accumulation in roots increased much more than that in leaves. SDS-PAGE analysis showed that the proteins changed more intensively in roots than did in leaves upon Cu stress. Two-dimensional gel electrophoresis (2-DE) and image analyses found that 45 protein spots were significantly changed in roots, but only six protein spots in leaves. The abundance of protein spots mostly showed temporal changes. MALDI-TOF MS and LTQ-ESI-MS/MS were used to identify the differently expressed protein spots. The identified root proteins were involved in various cellular processes such as signal transduction, regulation of transcription and translation, energy metabolism, regulation of redox homeostasis and cell defense. The leaf proteins were mainly degraded fragments of RuBisCo and antioxidative protein. The roles of these proteins in Cu tolerance/accumulation were discussed. The resulting differences in protein expression pattern suggested that redirection of root cellular metabolism and redox homeostasis might be important survival mechanisms of E. splendens upon Cu stress.

Pflugers Arch. 2009 Jul 22;
Gassanov N, Er F, Endres-Becker J, Wolny M, Schramm C, Hoppe UC

Thyroid hormone (TH) markedly modulates cardiovascular function and heart rate. The pacemaker current I(f) and encoding hyperpolarization-activated cation (HCN) genes have been identified as TH targets. To analyze the specific contribution and functional significance of thyroid receptor isoforms responsible for HCN gene transactivation, we generated transgenic neonatal rat cardiomyocytes with adenovirus-mediated overexpression of the thyroid receptors alpha1 (TRalpha1) and beta1 (TRbeta1), and analyzed native I(f) current and expression levels of the underlying molecular components HCN2 and HCN4. Initial results revealed that spontaneous beating activity was higher in TRalpha1- and lower in TRbeta1-expressing cardiomyocytes. This was associated with accelerated depolarization velocity and abbreviated action potential duration in cells overexpressing TRalpha1, while TRbeta1 suppressed phase 4 depolarization and prolonged action potentials. Consistently, TRalpha1-infected myocytes exhibited larger I(f) current densities along with increased HCN2 and HCN4 mRNA and protein levels. In contrast, HCN2 gene expression was not significantly affected by TRbeta1. TRbeta1 exclusively suppressed HCN4 transcription. T3 application led to significant effects only in controls and TRalpha1-infected cardiomyocytes; whereas, no ligand-dependent actions were observed in TRbeta1-expressing neonatal cardiomyocytes. Our results demonstrate that TRalpha1 and TRbeta1 divergently regulate cardiac pacing activity. TH-induced positive chronotropic effects are likely to be mediated by TRalpha1 through enhanced expression of I(f) pacemaker current and its underlying genes.

PLoS Pathog. 2009 Jul; 5(7): e1000526
Monaghan J, Xu F, Gao M, Zhao Q, Palma K, Long C, Chen S, Zhang Y, Li X

Plant Resistance (R) proteins play an integral role in defense against pathogen infection. A unique gain-of-function mutation in the R gene SNC1, snc1, results in constitutive activation of plant immune pathways and enhanced resistance against pathogen infection. We previously found that mutations in MOS4 suppress the autoimmune phenotypes of snc1, and that MOS4 is part of a nuclear complex called the MOS4-Associated Complex (MAC) along with the transcription factor AtCDC5 and the WD-40 protein PRL1. Here we report the immuno-affinity purification of the MAC using HA-tagged MOS4 followed by protein sequence analysis by mass spectrometry. A total of 24 MAC proteins were identified, 19 of which have predicted roles in RNA processing based on their homology to proteins in the Prp19-Complex, an evolutionarily conserved spliceosome-associated complex containing homologs of MOS4, AtCDC5, and PRL1. Among these were two highly similar U-box proteins with homology to the yeast and human E3 ubiquitin ligase Prp19, which we named MAC3A and MAC3B. MAC3B was recently shown to exhibit E3 ligase activity in vitro. Through reverse genetics analysis we show that MAC3A and MAC3B are functionally redundant and are required for basal and R protein-mediated resistance in Arabidopsis. Like mos4-1 and Atcdc5-1, mac3a mac3b suppresses snc1-mediated autoimmunity. MAC3 localizes to the nucleus and interacts with AtCDC5 in planta. Our results suggest that MAC3A and MAC3B are members of the MAC that function redundantly in the regulation of plant innate immunity.

PLoS Comput Biol. 2009 Jul; 5(7): e1000448
Pan Y, Nussinov R

p53-response elements (p53-REs) are organized as two repeats of a palindromic DNA segment spaced by 0 to 20 base pairs (bp). Several experiments indicate that in the vast majority of the human p53-REs there are no spacers between the two repeats; those with spacers, particularly with sizes beyond two nucleotides, are rare. This raises the question of what it indicates about the factors determining the p53-RE genomic organization. Clearly, given the double helical DNA conformation, the orientation of two p53 core domain dimers with respect to each other will vary depending on the spacer size: a small spacer of 0 to 2 bps will lead to the closest p53 dimer-dimer orientation; a 10-bp spacer will locate the p53 dimers on the same DNA face but necessitate DNA looping; while a 5-bp spacer will position the p53 dimers on opposite DNA faces. Here, via conformational analysis we show that when there are 0-2 bp spacers, p53-DNA binding is cooperative; however, cooperativity is greatly diminished when there are spacers with sizes beyond 2 bp. Cooperative binding is broadly recognized to be crucial for biological processes, including transcriptional regulation. Our results clearly indicate that cooperativity of the p53-DNA association dominates the genomic organization of the p53-REs, raising questions of the structural organization and functional roles of p53-REs with larger spacers. We further propose that a dynamic landscape scenario of p53 and p53-REs can better explain the selectivity of the degenerate p53-REs. Our conclusions bear on the evolutionary preference of the p53-RE organization and as such, are expected to have broad implications to other multimeric transcription factor response element organization.

EMBO J. 2009 Jul 23;
Luke B, Lingner J

Telomeres, the physical ends of eukaryotic chromosomes, consist of tandem arrays of short DNA repeats and a large set of specialized proteins. A recent analysis has identified telomeric repeat-containing RNA (TERRA), a large non-coding RNA in animals and fungi, which forms an integral component of telomeric heterochromatin. TERRA transcription occurs at most or all chromosome ends and it is regulated by RNA surveillance factors and in response to changes in telomere length. TERRA functions that are emerging suggest important roles in the regulation of telomerase and in orchestrating chromatin remodelling throughout development and cellular differentiation. The accumulation of TERRA at telomeres can also interfere with telomere replication, leading to a sudden loss of telomere tracts. Such a phenotype can be observed upon impairment of the RNA surveillance machinery or in cells from ICF (Immunodeficiency, Centromeric region instability, Facial anomalies) patients, in which TERRA is upregulated because of DNA methylation defects in the subtelomeric region. Thus, TERRA may mediate several crucial functions at the telomeres, a region of the genome that had been considered to be transcriptionally silent.

Science. 2009 Jul 23;
Nurieva RI, Chung Y, Martinez GJ, Yang XO, Tanaka S, Matskevitch TD, Wang YH, Dong C

A fundamental function of CD4(+) helper T (Th) cells is the regulation of B cell-mediated humoral immunity. Development of T follicular helper (Tfh) cells that provide help to B cells is mediated by the cytokines interleukin-6 and interleukin-21, but is independent of Th1, Th2, and Th17 effector cell lineages. Here, we characterize the function of Bcl6, a transcription factor selectively expressed in Tfh cells. Bcl6 expression is regulated by interleukin-6 and interleukin-21. Bcl6 overexpression induced Tfh-related gene expression and inhibited other Th lineage cell differentiation in a DNA binding-dependent manner. Moreover, Bcl6 deficiency in T cells resulted in impaired Tfh cell development, and germinal center reactions, and altered production of other effector T cells subsets. Our data thus illustrate that Bcl6 is required for programming of Tfh cell generation.

Am J Pathol. 2009 Jul 23;
Zhang L, Altuwaijri S, Deng F, Chen L, Lal P, Bhanot UK, Korets R, Wenske S, Lilja HG, Chang C, Scher HI, Gerald WL

Prostate cancers that progress during androgen-deprivation therapy often overexpress the androgen receptor (AR) and depend on AR signaling for growth. In most cases, increased AR expression occurs without gene amplification and may be due to altered transcriptional regulation. The transcription factor nuclear factor (NF)-kappa, which is implicated in tumorigenesis, functions as an important downstream substrate of mitogen-activated protein kinase, phosphatidylinositol 3-kinase, AKT, and protein kinase C and plays a role in other cancer-associated signaling pathways. NF-kappa is an important determinant of prostate cancer clinical biology, and therefore we investigated its role in the regulation of AR expression. We found that NF-kappa expression in prostate cancer cells significantly increased AR mRNA and protein levels, AR transactivation activity, serum prostate-specific antigen levels, and cell proliferation. NF-kappa inhibitors decrease AR expression levels, prostate-specific antigen secretion, and proliferation of prostate cancer cells in vitro. Furthermore, inhibitors of NF-kappa demonstrated anti-tumor activity in androgen deprivation-resistant prostate cancer xenografts. In addition, levels of both NF-kappa and AR were strongly correlated in human prostate cancer. Our data suggest that NF-kappa can regulate AR expression in prostate cancer and that NF-kappa inhibitors may have therapeutic potential.

Endocr Relat Cancer. 2009 Jul 23;
Welboren WJ, Sweep FC, Span P, Stunnenberg H

The estrogen receptor alpha (ERalpha) is a ligand dependent transcription factor that regulates a large number of genes in many different target tissues and is important in the development and progression of breast cancer. ERalpha-mediated transcription is a complex process regulated at many different levels. The interplay between ligand, receptor, DNA sequence, cofactors, chromatin context and post-translational modifications culminates in transcriptional regulation by ERalpha. Recent technological advances have allowed the identification of ERalpha target genes on a genome-wide scale. In this review we provide an overview of the progress made in our understanding of the different levels of regulation mediated by ERalpha. We discuss the recent advances in the identification of the ERalpha binding sites and target gene network and their clinical applications.

Mol Endocrinol. 2009 Jul 23;
Dobkin-Bekman M, Naidich M, Rahamim L, Przedecki F, Almog T, Lim S, Melamed P, Liu P, Wohland T, Yao Z, Seger R, Naor Z

Most receptor tyrosine kinases (RTKs) and G-protein coupled receptors (GPCRs) operate via a limited number of MAPK cascades, but still exert diverse functions and therefore signal specificity remains an enigma. Also, most GPCR ligands utilize families of receptors for mediation of diverse biological actions; however the mammalian type I GnRH receptor (GnRHR) seems to be the sole receptor mediating GnRH-induced gonadotropin synthesis and release. Signaling complexes associated with GPCRs may thus provide the means for signal specificity. Here we describe a signaling complex associated with the GnRHR, which is a unique GPCR lacking a C-terminal tail. Unlike other GPCRs, this signaling complex is pre-formed and exposure of LbetaT2 gonadotropes to GnRH induces its dynamic rearrangement. The signaling complex includes c-Src, PKC delta, epsilon and alpha, Ras, MEK1/2, ERK1/2, tubulin, FAK, paxillin, vinculin, caveolin-1, KSR-1 and the GnRHR. Exposure to GnRH (5 min) causes MEK1/2, ERK1/2, tubulin, vinculin and the GnRHR to detach from c-Src, but they re-associate within 30 min. On the other hand, FAK, paxillin, the PKCs and caveolin-1 stay bound to c-Src, while KSR-1 appears in the complex only 30 min after GnRH stimulation. GnRH was found to activate ERK1/2 in the complex in a c-Src-dependent manner and the activated ERK1/2 subsequently phosphorylates FAK and paxillin. In parallel, caveolin-1, FAK, vinculin and paxillin are phosphorylated on Tyr residues apparently by GnRH-activated c-Src. RTKs and GPCRs translocate ERK1/2 to the nucleus to phosphorylate and activate transcription factors. We therefore propose that the role of the multi-protein signaling complex is to sequester a cytosolic pool of activated ERK1/2 to phosphorylate FAK and paxillin at focal adhesions.

Mol Endocrinol. 2009 Jul 23;
Chan IH, Privalsky ML

Thyroid hormone receptors (TRs) are hormone-regulated transcription factors that control multiple aspects of physiology and development. TRs are expressed in vertebrates as a series of distinct isoforms that exert distinct biological roles. We wished to determine if the two most widely expressed isoforms, TRalpha1 and TRbeta1, exert their different biological effects by regulating different sets of target genes. Using stably transformed HepG2 cells and a microarray analysis, we were able to demonstrate that TRalpha1 and TRbeta1 regulate a largely overlapping repertoire of target genes in response to T3 hormone. However, these two isoforms display very different transcriptional properties on each individual target gene, ranging from a much greater T3-mediated regulation by TRalpha1 than by TRbeta1, to near equal regulation by both isoforms. We also identified TRalpha1 and TRbeta1 target genes that were regulated by these receptors in a hormone-independent fashion. We suggest that it is this gene-specific, isoform-specific amplitude of transcriptional regulation that is the likely basis for the appearance and maintenance of TRalpha1 and TRbeta1 over evolutionary time. In essence, TRalpha1 and TRbeta1 adjust the magnitude of the transcriptional response at different target genes to different levels; by altering the ratio of these isoforms in different tissues or at different developmental times, the intensity of T3 response can be individually tailored to different physiological and developmental requirements.

J Exp Bot. 2009 Jul 23;
Frank G, Pressman E, Ophir R, Althan L, Shaked R, Freedman M, Shen S, Firon N

Above-optimal temperatures reduce yield in tomato largely because of the high heat stress (HS) sensitivity of the developing pollen grains. The high temperature response, especially at this most HS-sensitive stage of the plant, is poorly understood. To obtain an overview of molecular mechanisms underlying the HS response (HSR) of microspores, a detailed transcriptomic analysis of heat-stressed maturing tomato microspores was carried out using a combination of Affymetrix Tomato Genome Array and cDNA-amplified fragment length polymorphism (AFLP) techniques. The results were corroborated by reverse transcription-PCR (RT-PCR) and immunoblot analyses. The data obtained reveal the involvement of specific members of the small heat shock protein (HSP) gene family, HSP70 and HSP90, in addition to the HS transcription factors A2 (HSFA2) and HSFA3, as well as factors other than the classical HS-responsive genes. The results also indicate HS regulation of reactive oxygen species (ROS) scavengers, sugars, plant hormones, and regulatory genes that were previously implicated in other types of stress. The use of cDNA-AFLP enabled the detection of genes representing pollen-specific functions that are missing from the tomato Affymetrix chip, such as those involved in vesicle-mediated transport and a pollen-specific, calcium-dependent protein kinase (CDPK2). For several genes, including LeHSFA2, LeHSP17.4-CII, as well as homologues of LeHSP90 and AtVAMP725, higher basal expression levels were detected in microspores of cv. Hazera 3042 (a heat-tolerant cultivar) compared with microspores of cv. Hazera 3017 (a heat-sensitive cultivar), marking these genes as candidates for taking part in microspore thermotolerance. This work provides a comprehensive analysis of the molecular events underlying the HSR of maturing microspores of a crop plant, tomato.

Cancer Lett. 2009 Jul 21;
Zhang HY, Sun H

The transcription factor Forkhead Box P3 (Foxp3) has been shown to play important roles in the occurring of regulatory T cells (Tregs). Limited evidence indicated that it was also expressed in tissues other than thymus and spleen, while, very recently, it was identified as a suppressor gene in breast cancer. However, the precise role and molecular mechanism of the action of Foxp3 in ovarian cancer remained unclear. To elucidate the function of Foxp3, we examined the expression of Foxp3 in ovarian cancerous cells and the consequences of up-regulation of Foxp3 in epithelial ovarian cancer cell lines, respectively. By multiple cellular and molecular approaches such as gene transfection, CCK-8 assay, flow cytometry, RT-PCR, in-cell western, wound healing assay, and invasion assay, we found that Foxp3 was weakly/no expressed in ovarian cancerous cells. Up-regulation of Foxp3 inhibited cell proliferation, decreased cell migration, and reduced cell invasion. Compared with control, Foxp3 up-regulated cells showed decreased expression of Ki-67 and cyclin-dependent kinases (CDKs). Moreover, up-regulation of Foxp3 reduced the expression of matrix metalloproteinase-2 (MMP-2) and urokinase-type plasminogen activator (uPA), resulting in the inhibition of cell migration and invasion. In addition, Foxp3 up-regulation inhibited the activation of mammalian target of rapamycin (mTOR) and NF-kappaB signaling. These findings suggested that up-regulation of Foxp3 could be a novel approach for inhibiting ovarian cancer progression.

J Plant Physiol. 2009 Jul 21;
Jia X, Ren L, Chen QJ, Li R, Tang G

MicroRNAs (miRNAs) play vital roles in down-regulating gene expression at the post-transcriptional level. A set of 24 UV-B stress-responsive miRNAs (13 up-regulated and 11 down-regulated) was identified in Populus tremula plantlet by expression profiling with our in-house miRNA filter array. Six of the UV-B-responsive miRNA and their corresponding target genes were verified for their expressions by RNA blotting and quantitative reverse transcription PCR (qRT-PCR), respectively. The predicted target genes for these miRNAs encode diverse proteins including transcription factors and phytohormone signal-related proteins. Promoter analysis of the UV-B-responsive miRNAs revealed the presence of many light-relevant cis-elements. However, these cis-elements were not necessarily specific to the promoters of UV-responsive miRNAs, indicating that other machinery may be involved in the regulation of UV-responsive miRNAs. Finally, a model was developed to describe the potential regulatory networks mediated by the UV-B-responsive miRNAs in P. tremula. These results provide new insights into the understanding of miRNAs as ubiquitous regulators in plant response to UV-B and other stresses.

Dev Biol. 2009 Jul 20;
Roush SF, Slack FJ

The let-7 family of microRNAs (miRNAs) are important regulators of developmental timing and cell differentiation and are often misexpressed in human cancer. In C. elegans, let-7 controls cell fate transitions from larval stage 4 (L4) to adulthood by post-transcriptionally down-regulating lineage-abnormal 41 (lin-41) and hunchback-like 1 (hbl-1). Primary let-7 (pri-let-7) transcripts are up-regulated in the L3, yet little is known about what controls this transcriptional up-regulation. We sought factors that either turn on let-7 transcription or keep it repressed until the correct time. Here we report that one of let-7's targets, the transcription factor Hunchback-like 1 (HBL-1), is responsible for inhibiting the transcription of let-7 in specific tissues until the L3. hbl-1 is a known developmental timing regulator and inhibits adult development in larval stages. Therefore, one important function of HBL-1 in maintaining larval stage fates is inhibition of let-7. Indeed, our results reveal let-7 as the first known target of the HBL-1 transcription factor in C. elegans and suggest a negative feedback loop mechanism for let-7 and HBL-1 regulation.

J Cell Mol Med. 2009 Jul 20;
Aarts M, Te Riele H

ABSTRACT Gene targeting by single-stranded oligodeoxyribonucleotides (ssODN) is emerging as a powerful tool for the introduction of subtle gene modifications in mouse embryonic stem (ES) cells and the generation of mutant mice. Here, we have studied the role of ssODN composition, transcription and replication of the target locus, and DNA repair pathways to gain more insight into the parameters governing ssODN-mediated gene targeting in mouse ES cells. We demonstrated that unmodified ssODN of 35-40 nt were most efficient in correcting a chromosomally integrated mutant neomycin reporter gene. Addition of chemical modifications did not further enhance the efficacy of these ssODN. The observed strand bias was not affected by transcriptional activity and may rather be caused by the different accessibility of the DNA strands during DNA replication. Consistently, targeting frequencies were enhanced when cells were treated with hydroxyurea to reduce the rate of replication fork progression. Transient down regulation of various DNA repair genes by RNAi had no effect on the targeting frequency. Taken together, our data suggest that ssODN-mediated gene targeting occurs within the context of a replication fork. This implies that any given genomic sequence, irrespective of transcriptional status, should be amenable to ssODN-mediated gene targeting. The ability of ES cells to differentiate into various cell types after ssODN-mediated gene targeting may offer opportunities for future therapeutic applications.

Drug Metab Rev. 2009 Jul 23;
Monostory K, Pascussi JM, Kóbori L, Dvorak Z

Aryl hydrocarbon receptor (AhR) is primarily involved in the transcriptional regulation of CYP1A enzymes, which are the main catalysts of the metabolic activation or inactivation of numerous procarcinogens, carcinogens, environmental toxicants, or therapeutic agents. Several endogenous factors, including hormones, can modify CYP1A induction, directly influencing CYP1A gene expression or AhR function or indirectly via crosstalks with other transcription factors. In this article, we summarize the current knowledge about the role of hormones (i.e., glucocorticoids, estrogens, progesterone, androgens, insulin, and glucagon) and hormone receptors as well as other essential factors (e.g., vitamin D, retinoids) in the modulation of CYP1A expression.

Proteins. 2009 Jun 19;
Burendahl S, Danciulescu C, Nilsson L

The estrogen receptor (ER) belongs to the nuclear receptor superfamily, whose members regulate important cellular events like development and metabolism. The ER functions as a transcription regulator and can be activated on ligand binding. Consequently, ligand binding and unbinding constitute fundamental processes in the regulation of genes. Even though both biochemical and structural data of ER are available, the actual mechanism of the ligand binding/unbinding remains elusive. We have performed computational studies on the unbinding mechanism of ERalpha and ERbeta, in the presence of cofactors and with ligands ranging from agonist to a full antagonist. Our results show that agonists or selective ER modulators can dissociate from the receptor through multiple pathways with minor effect on the receptor structure, whereas an antagonist requires larger conformational changes. Furthermore, a specific receptor/ligand combination can exhibit a pathway preference depending on character and conformation of both parts. Hence, it is possible that the binding/unbinding mechanism can explain ligand subtype specificity and thus have an impact in drug discovery. Proteins 2009. (c) 2009 Wiley-Liss, Inc.

J Cell Physiol. 2009 Jul 22;
Charles S, Ammosova T, Cardenas J, Foster A, Rotimi J, Jerebtsova M, Ayodeji AA, Niu X, Ray PE, Gordeuk VR, Kashanchi F, Nekhai S

HIV transcription is induced by the HIV-1 Tat protein, in concert with cellular co-factors including CDK9, CDK2, NF-kappaB, and others. The cells of most of the body's organs are exposed to approximately 3-6% oxygen, but most in vitro studies of HIV replication are conducted at 21% oxygen. We hypothesized that activities of host cell factors involved in HIV-1 replication may differ at 3% versus 21% O(2), and that such differences may affect HIV-1 replication. Here we show that Tat-induced HIV-1 transcription was reduced at 3% O(2) compared to 21% O(2). HIV-1 replication was also reduced in acutely or chronically infected cells cultured at 3% O(2) compared to 21% O(2). This reduction was not due the decreased cell growth or increased cellular toxicity and also not due to the induction of hypoxic response. At 3% O(2), the activity of CDK9/cyclin T1 was inhibited and Sp1 activity was reduced, whereas the activity of other host cell factors such as CDK2 or NF-kappaB was not affected. CDK9-specific inhibitor ARC was much less efficient at 3% compared to 21% O(2) and also expression of CDK9/cyclin T1-dependent IkappaB inhibitor alpha was repressed. Our results suggest that lower HIV-1 transcription at 3% O(2) compared to 21% O(2) may be mediated by lower activity of CDK9/cyclin T1 and Sp1 at 3% O(2) and that additional host cell factors such as CDK2 and NF-kappaB might be major regulators of HIV-1 transcription at low O(2) concentrations. J. Cell. Physiol. (c) 2009 Wiley-Liss, Inc.

J Cell Physiol. 2009 Jul 22;
Wang H, Yu YQ, Liao WJ, Wang ZR, Lv YJ, Zhang YG, Gao TW

Regulations of intracellular protein kinase C (PKC) on carbachol (CCh)-induced intracellular calcium ([Ca(2+)]i) responses were investigated in different stages of melanoma cells. We found that CCh (1 mM) significantly increased [Ca(2+)]i with 6-, 4-, 4-, and 25-folds intensities in WM793B, 451Lu, SK-MEL-5, and A2058 melanoma cells, respectively. Pretreatment of phorbol 12, 13-dibutyrate (PDBu, 2 microM), an activator of intracellular PKC, significantly suppressed CCh-induced peak reactions in WM793B, SK-MEL-5, and A2058 cells. RT-PCR data showed that mRNA levels of PKCalpha were 12-, 4-, 6-, and 0.9-folds higher in above four melanoma cells. Short interfering RNA (siRNA) targeting to PKCalpha in WM793B cells enhanced CCh-induced peak calcium reactions. Present data indicated that CCh-induced [Ca(2+)]i responses were dynamically changed in different stages of melanoma progression. Moreover, intracellular PKCalpha activated by exogenous agonist and expressed through endogenous gene transcription negatively regulated CCh-induced calcium responses. The functional analysis on the relationship between CCh-induced calcium response and endogenous PKCalpha expression might be helpful to predict the development of melanoma. J. Cell. Physiol. (c) 2009 Wiley-Liss, Inc.

J Cell Physiol. 2009 Jul 22;
Gordon GM, Ledee DR, Feuer WJ, Fini ME

Matrix metalloproteinase-9 (MMP-9) is a well-known regulator and effecter of many cellular processes including wound healing. In the cornea, either too much or too little MMP-9 can be detrimental to overall wound repair. We investigated the secreted factors as well as the intracellular signaling pathways and the promoter sequences that mediate this regulation. Primary culture rabbit corneal epithelial cells were treated with various cytokines alone or in different combinations and MMP-9 induction was assessed by gel zymography. Pharmacological inhibitors were used to determine the intracellular signaling pathways induced by the cytokines tested and deletion promoter constructs were created to determine the regions of the MMP-9 promoter involved in the cytokine regulation, thereby assessing the exact transcription factors binding the MMP-9 promoter. We found that two cytokine families, transforming growth factor beta (TGF-beta) and interleukin 1 (IL-1), act additively in an isoform non-specific manner to induce MMP-9 in this cell type. Our data suggest TGF-beta mediated MMP-9 induction may be regulated by the NF-kappaB, Smad3, and JNK pathways, whereas the IL-1beta mediated induction may be regulated by the NF-kappaB and p38 pathways. Inhibition of the p38, NF-kappaB, or JNK pathways significantly reduced, but did not abrogate, basal MMP-9 levels. Inhibition of the ERK pathway did not have an effect on MMP-9 mediated expression in either the treated or untreated co-transfected cells. J. Cell. Physiol. (c) 2009 Wiley-Liss, Inc.