Complex regulation of membrane-type matrix metalloproteinase expression and matrix metalloproteinase-2 activation by concanavalin A in MDA-MB-231 human breast cancer cells

Matrix Metalloproteinase-2 (MMP-2) is secreted as a zymogen, the activation of which has been associated with metastatic progression in human breast cancer (HBC). Concanavalin A (Con A) has been found to induce activation of MMP-2 in invasive HBC cell lines. Con A effects on the expression of mRNA for membrane-type matrix metalloproteinase (MT-MMP), a newly described cell surface-associated MMP, showed a close temporal correlation with induction of MMP-2 activation. It is surprising that MT-MMP mRNA is constitutively present in the uninduced MDA-MB-231 cell, despite a lack of MMP-2 activation. We have used actinomycin D to demonstrate a partial requirement for de novo gene expression in the induction of MMP-2 activation by Con A in MDA-MB-231 HBC cells. Furthermore, this transcriptional response to Con A appeared to require the continued presence of Con A for its manifestation. The nontranscriptional component of the Con A induction manifests rapidly, is quite substantial, and persists strongly despite actinomycin D abrogation of both constitutive and Con A-induced MT-MMP. Cycloheximide analyses suggest that protein synthesis may be involved in this rapid transcription-independent response. These studies suggest that Con A induces MMP-2-activation in part by up-regulation of MT-MMP expression but has a more complicated mode of action, involving additional nontranscriptional effects, which apparently require protein synthesis.

Functional role for senataxin, defective in ataxia oculomotor apraxia type 2, in transcriptional regulation

Ataxia oculomotor apraxia type 2 (AOA2) is an autosomal recessive neurodegenerative disorder characterized by cerebellar ataxia and oculomotor apraxia. The gene mutated in AOA2, SETX, encodes senataxin, a putative DNA/RNA helicase which shares high homology to the yeast Sen1p protein and has been shown to play a role in the response to oxidative stress. To investigate further the function of senataxin, we identified novel senataxin-interacting proteins, the majority of which are involved in transcription and RNA processing, including RNA polymerase II. Binding of RNA polymerase II to candidate genes was significantly reduced in senataxin deficient cells and this was accompanied by decreased transcription of these genes, suggesting a role for senataxin in the regulation/modulation of transcription. RNA polymerase II-dependent transcription termination was defective in cells depleted of senataxin in keeping with the observed interaction of senataxin with poly(A) binding proteins 1 and 2. Splicing efficiency of specific mRNAs and alternate splice-site selection of both endogenous genes and artificial minigenes were altered in senataxin depleted cells. These data suggest that senataxin, similar to its yeast homolog Sen1p, plays a role in coordinating transcriptional events, in addition to its role in DNA repair.

Transglutaminase-2 Regulation by Arecoline in Gingival Fibroblasts

Transglutaminase-2 (TGM-2) stabilizes extracellular matrix (ECM) proteins by cross-linking and has been implicated in several fibrotic disorders. Arecoline present in betel quid has been proposed as one of the causative factors for oral submucous fibrosis (OSMF). Hence, we hypothesize that arecoline may regulate TGM-2 and may have a role in the pathogenesis of OSMF. The expression of TGM-2 was studied in OSMF tissues by real-time RT-PCR analysis, and significant overexpression was observed in most OSMF tissues (P = 0.0112) compared with normal tissues. Arecoline induced TGM-2 mRNA and protein expression as well as TGM-2 activity in human gingival fibroblast cells. The addition of methocramine hemihydrate (M-2 muscarinic acetylcholine receptor selective antagonist) or 8'-bromo-cAMP abolished arecoline-mediated TGM-2 induction, suggesting a role for M-2 muscarinic acid receptor and a repressor role for cAMP. Our study provides evidence for TGM-2 overexpression in OSMF and its regulation by arecoline in oral fibroblasts.

Genome-wide association and genetic functional studies identify autism susceptibility candidate 2 gene (AUTS2) in the regulation of alcohol consumption

Alcohol consumption is a moderately heritable trait, but the genetic basis in humans is largely unknown, despite its clinical and societal importance. We report a genome-wide association study meta-analysis of approximately 2.5 million directly genotyped or imputed SNPs with alcohol consumption (gram per day per kilogram body weight) among 12 population-based samples of European ancestry, comprising 26,316 individuals, with replication genotyping in an additional 21,185 individuals. SNP rs6943555 in autism susceptibility candidate 2 gene (AUTS2) was associated with alcohol consumption at genome-wide significance (P = 4 x 10(-8) to P = 4 x 10(-9)). We found a genotype-specific expression of AUTS2 in 96 human prefrontal cortex samples (P = 0.026) and significant (P < 0.017) differences in expression of AUTS2 in whole-brain extracts of mice selected for differences in voluntary alcohol consumption. Down-regulation of an AUTS2 homolog caused reduced alcohol sensitivity in Drosophila (P < 0.001). Our finding of a regulator of alcohol consumption adds knowledge to our understanding of genetic mechanisms influencing alcohol drinking behavior.

Functional regulation of the Neurofibromatosis 2 tumor suppressor merlin

Neurofibromatosis 2 (NF2) is an autosomal dominant disorder manifested by the formation of multiple benign tumors of the nervous system. Affected individuals typically develop bilateral vestibular schwannomas which lead to deafness and balance disorders. The syndrome is caused by inactivation of the NF2 tumor suppressor gene, and mutation or loss of the NF2 product, merlin, is sufficient for tumorigenesis in both hereditary and sporadic NF2-associated tumors. Merlin belongs to the band 4.1 superfamily of cytoskeletal proteins, which also contain the related ezrin, radixin, and moesin (ERM) proteins. The ERM members provide a link between the cell cytoskeleton and membrane by connecting membrane-associated proteins to actin filaments. By stabilizing complexes in the cell cortex, the ERMs modulate morphology, growth, and migration of cells. Despite their structural homology, overlapping subcellular distribution, direct molecular association, and partial overlap of molecular interactions, merlin and ezrin exert opposite effects on cell proliferation. Merlin suppresses cell proliferation, whereas ezrin expression is linked to oncogenic activity. We hypothesized that the regions which differ between the proteins might explain merlin s specificity as a tumor suppressor. We therefore analyzed the regions, which are most diverse between merlin and ezrin; the N-terminal tail and the C-terminus. To determine the properties of the C-terminal region, we studied the two most predominant merlin isoforms together with truncation variants similar to those found in patients. We also focused on the evolutionally conserved C-terminal residues, E545-E547, that harbor disease causing mutations in its corresponding DNA sequence. In addition to inhibiting cell proliferation, merlin regulates cytoskeletal organization. The morphogenic properties of merlin may play a role in tumor suppression, since patient-derived tumor cells demonstrate cytoskeletal abnormalities. We analyzed the mechanisms of merlin-induced extension formation and determined that the C-terminal region of amino acids 538-568 is particularly important for the morphogenic activity. We also characterized the role of C-terminal merlin residues in the regulation of proliferation, phosphorylation, and intramolecular associations. In contrast to previous reports, we demonstrated that both merlin isoforms are able to suppress cell proliferation, whereas C-terminally mutated merlin constructs showed reduced growth inhibition. Phosphorylation serves as a mechanism to regulate the tumor suppressive activity of merlin. The C-terminal serine 518 is phosphorylated in response to both p21-activated kinase (PAK) and protein kinase A (PKA), which inactivates the growth inhibitory function of merlin. However, at least three differentially phosphorylated forms of the protein exist. In this study we demonstrated that also the N-terminus of merlin is phosphorylated by AGC kinases, and that both PKA and Akt phosphorylate merlin at serine 10 (S10). We evaluated the impact of this N-terminal tail phosphorylation, and showed that the phosphorylation state of S10 is an important regulator of merlin s ability to modulate cytoskeletal organization but also regulates the stability of the protein. In summary, this study describes the functional effect of merlin specific regions. We demonstrate that both S10 in the N-terminal tail and residues E545-E547 in the C-terminus are essential for merlin activity and function.

Regulation of Chemokines, CCL3 and CCL4, by interferon gamma and Nitric oxide synthase 2 in mouse macrophages and during Salmonella enterica Serovar Typhimurium infection

Background. Interferon gamma (IFN-gamma) increases the expression of multiple genes and responses; however, the mechanisms by which IFN-gamma downmodulates cellular responses is not well understood. In this study, the repression of CCL3 and CCL4 by IFN-gamma and nitric oxide synthase 2 (NOS2) in macrophages and upon Salmonella typhimurium infection of mice was investigated. Methods. Small molecule regulators and adherent peritoneal exudates cells (A-PECs) from Nos2(-/-)mice were used to identify the contribution of signaling molecules during IFN-gamma-mediated in vitro regulation of CCL3, CCL4, and CXCL10. In addition, infection of bone marrow-derived macrophages (BMDMs) and mice (C57BL/6, Ifn-gamma(-/), and Nos2(-/-)) with S. typhimurium were used to gain an understanding of the in vivo regulation of these chemokines. Results. IFN-gamma repressed CCL3 and CCL4 in a signal transducer and activator of transcription 1 (STAT1)-NOS2-p38 mitogen activated protein kinase (p38MAPK)-activating transcription factor 3 (ATF3) dependent pathway in A-PECs. Also, during intracellular replication of S. typhimurium in BMDMs, IFN-gamma and NOS2 repressed CCL3 and CCL4 production. The physiological roles of these observations were revealed during oral infection of mice with S. typhimurium, wherein endogenous IFN-gamma and NOS2 enhanced serum amounts of tumor necrosis factor alpha and CXCL10 but repressed CCL3 and CCL4. Conclusions. This study sheds novel mechanistic insight on the regulation of CCL3 and CCL4 in mouse macrophages and during S. typhimurium oral infection.

Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain

Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (Al-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the Al-2 synthase was cloned from TX1 and designated luxS(Et). LuxS(Et) was able to complement the Al-2 mutant phenotype of Escherichia coli strain DH5 alpha. Expression Of luxS(Et) correlated with Al-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS(Et) expression. Overexpression of luxS(Et) enhanced Al-2 activity in TX1, whereas disruption of luxS(Et) expression by antisense RNA interference (i) reduced the level of Al-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous Al-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the Al-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS(Et) is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/Al-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.

Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain

Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (Al-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the Al-2 synthase was cloned from TX1 and designated luxS(Et). LuxS(Et) was able to complement the Al-2 mutant phenotype of Escherichia coli strain DH5 alpha. Expression Of luxS(Et) correlated with Al-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS(Et) expression. Overexpression of luxS(Et) enhanced Al-2 activity in TX1, whereas disruption of luxS(Et) expression by antisense RNA interference (i) reduced the level of Al-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous Al-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the Al-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS(Et) is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/Al-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.

Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/beta 2-adrenergic receptor.

The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (beta ARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the beta AR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta 2AR has a C terminus containing 11 serine and threonine residues representing potential sites for beta AR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta 3AR, and they are in a nonfavorable context. The beta 3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta 2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta 3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta 3/beta 2AR which comprised the beta 3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta 2AR. When cells expressing this chimeric beta 3/beta 2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta 2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta 3AR, even when beta AR kinase was overexpressed. In contrast, the chimeric beta 3/beta 2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In addition to conferring functional desensitization and phosphorylation to the beta 3AR, the C-terminal tail of the beta 2AR also conferred agonist-promoted sequestration and long-term receptor down-regulation.

Involvement of tyrosine residues located in the carboxyl tail of the human beta 2-adrenergic receptor in agonist-induced down-regulation of the receptor.

Chronic exposure of various cell types to adrenergic agonists leads to a decrease in cell surface beta 2-adrenergic receptor (beta 2AR) number. Sequestration of the receptor away from the cell surface as well as a down-regulation of the total number of cellular receptors are believed to contribute to this agonist-mediated regulation of receptor number. However, the molecular mechanisms underlying these phenomena are not well characterized. Recently, tyrosine residues located in the cytoplasmic tails of several membrane receptors, such as the low density lipoprotein and mannose-6-phosphate receptors, have been suggested as playing an important role in the agonist-induced internalization of these receptors. Accordingly, we assessed the potential role of two tyrosine residues in the carboxyl tail of the human beta 2AR in agonist-induced sequestration and down-regulation of the receptor. Tyr-350 and Tyr-354 of the human beta 2AR were replaced with alanine residues by site-directed mutagenesis and both wild-type and mutant beta 2AR were stably expressed in transformed Chinese hamster fibroblasts. The mutation dramatically decreased the ability of the beta 2AR to undergo isoproterenol-induced down-regulation. However, the substitution of Tyr-350 and Tyr-354 did not affect agonist-induced sequestration of the receptor. These results suggest that tyrosine residues in the cytoplasmic tail of human beta 2AR are crucial determinants involved in its down-regulation.

Small Proline Rich Protein-2 Expression and Regulation in the Caco-2 model of Intestinal Epithelial Differentiation along the Crypt-Villus Axis

Small proline-rich protein-2 (SPRR2) functions as a determinant of flexibility and permeability in the mature cornified envelope of the skin. SPRR2 is strongly upregulated by the commensal flora and may mediate signaling to differentiated epithelia of the small intestine and colon. Yet, SPRR2 function in the GI tract is largely unexplored. Using the Caco-2 model of intestinal epithelial differentiation along the crypt-villus axis, we hypothesized that SPRR2 would be preferentially expressed in post-confluent differentiated Caco-2 cells and examined SPRR2 regulation by the protein kinase A pathway (PKA) and short chain fatty acids (SCFAs). Differentiation-dependent SPRR2 expression was examined in cytoskeletal-, membrane-, and nuclear-enriched fractions by immunoblotting and confocal immunofluorescence. We studied the effect of SCFAs, known inducers of differentiation, on SPRR2 expression in pre-confluent undifferentiated Caco-2 cells and explored potential mechanisms involved in this induction using MAP kinase inhibitors. SPRR2 expression was also compared between HIEC crypt cells and 16 to 20 week primary fetal villus cells as well as in different segments in mouse small intestine and colon. We determined if SPRR2 is increased by gram negative bacteria such as S. typhimurium. SPRR2 expression increased in a differentiation-dependent manner in Caco-2 cells and was present in human fetal epithelial villus cells but absent in HIEC crypt cells. Differentiation-induced SPRR2 was down-regulated by 8-Br-cAMP as well as by forskolin/IBMX co-treatment. SPRR2 was predominantly cytoplasmic and did not accumulate in Triton X-100-insoluble cytoskeletal fractions. SPRR2 was present in the membrane- and nuclear-enriched fractions and demonstrated co-localization with F-actin at the apical actin ring. No induction was seen with the specific HDAC inhibitor trichostatin A, while SCFAs and the HDAC inhibitor SBHA all induced SPRR2. SCFA responses were inhibited by MAP kinase inhibitors SB203580 and U0126, thus suggesting that the SCFA effect may be mediated by orphan G-protein receptors GPR41 and GPR43. S. typhimurium induced SPRR2 in undifferentiated cells. We conclude that SPRR2 protein expression is associated with differentiated epithelia and is regulated by PKA signaling and by by-products of the bowel flora. This is the first report to establish an in vitro model to study the physiology and regulation of SPRR2.

Down-regulation of Rac Activity during beta 2 Integrin-mediated Adhesion of Human Neutrophils

In human neutrophils, beta2 integrin engagement mediated a decrease in GTP-bound Rac1 and Rac2. Pretreatment of neutrophils with LY294002 or PP1 (inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases, respectively) partly reversed the beta2 integrin-induced down-regulation of Rac activities. In contrast, beta2 integrins induced stimulation of Cdc42 that was independent of Src family members. The PI 3-kinase dependency of beta2 integrin-mediated decrease in GTP-bound Rac could be explained by an enhanced Rac-GAP activity, since this activity was blocked by LY204002, whereas PP1 only had a minor effect. The fact that only Rac1 but not Rac2 (the dominating Rac) redistributed to the detergent-insoluble fraction and that it was independent of GTP loading excludes the possibility that down-regulation of Rac activities was due to depletion of GTP-bound Rac from the detergent-soluble fraction. The beta2 integrin-triggered relocalization of Rac1 to the cytoskeleton was enabled by a PI 3-kinase-induced dissociation of Rac1 from LyGDI. The dissociations of Rac1 and Rac2 from LyGDI also explained the PI 3-kinase-dependent translocations of Rac GTPases to the plasma membrane. However, these accumulations of Rac in the membrane, as well as that of p47phox and p67phox, were also regulated by Src tyrosine kinases. Inasmuch as Rac GTPases are part of the NADPH oxidase and the respiratory burst is elicited in neutrophils adherent by beta2 integrins, our results indicate that activation of the NADPH oxidase does not depend on the levels of Rac-GTP but instead requires a beta2 integrin-induced targeting of the Rac GTPases as well as p47phox and p67phox to the plasma membrane.

CDK11(p58) protein kinase activity is associated with Bcl-2 down-regulation in pro-apoptosis pathway.

CDK11(p58), a G2/M-specific protein kinase, has been shown to be associated with apoptosis in many cell lines, with largely unknown mechanisms. Our previous study proved that CDK11(p58)-enhanced cycloheximide (CHX)-induced apoptosis in SMMC-7721 hepatocarcinoma cells. Here we report for the first time that ectopic expression of CDK11(p58) down-regulates Bcl-2 expression and its Ser70, Ser87 phosphorylation in CHX-induced apoptosis in SMMC-7721 cells. Overexpression of Bcl-2 counteracts the pro-apoptotic activity of CDK11(p58). Furthermore, we confirm that the kinase activity of CDK11(p58) is essential to the down-regulation of Bcl-2 as well as apoptosis. Taken together, these results demonstrate that CDK11(p58) down-regulates Bcl-2 in pro-apoptosis pathway depending on its kinase activity, which elicits survival signal in hepatocarcinoma cells.