The plasmodium receptor for activated C kinase protein inhibits Ca(2+) signaling in mammalian cells

Plasmodium falciparum, the most lethal malarial parasite, expresses an ortholog for the protein kinase C (PKC) activator RACK1. However, PKC has not been identified in this parasite, and the mammalian RACK1 can interact with the inositol 1,4,5-trisphosphate receptor (InsP3R). Therefore we investigated whether the Plasmodium ortholog PfRACK also can affect InsP3R-mediated Ca(2+) signaling in mammalian cells. GFP-tagged PfRACK and endogenous RACK1 were expressed in a similar distribution within cells. PfRACK inhibited agonist-induced Ca(2+) signals in cells expressing each isoform of the InsP3R, and this effect persisted when expression of endogenous RACK1 was reduced by siRNA. PfRACK also inhibited Ca(2+) signals induced by photorelease of caged InsP3. These findings provide evidence that PfRACK directly inhibits InsP3-mediated Ca(2+) signaling in mammalian cells. Interference with host cell signaling pathways to subvert the host intracellular milieu may be an important mechanism for parasite survival. (C) 2009 Elsevier Inc. All rights reserved.

Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells

Carraro-Lacroix LR, Malnic G, Girardi AC. Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells. Am J Physiol Renal Physiol 297: F1647-F1655, 2009. First published September 23, 2009; doi:10.1152/ajprenal.00082.2009.-The gut incretin hormone glucagon-like peptide 1 (GLP-1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP-1 has been shown to have natriuretic actions paralleled by a diminished proton secretion. We therefore studied the role of the GLP-1 receptor agonist exendin-4 in modulating the activity of Na(+)/H(+) exchanger NHE3 in LLC-PK(1) cells. We found that NHE3-mediated Na(+)-dependent intracellular pH (pH(i)) recovery decreased similar to 50% after 30-min treatment with 1 nM exendin-4. Pharmacological inhibitors and cAMP analogs that selectively activate protein kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC) demonstrated that regulation of NHE3 activity by exendin-4 requires activation of both cAMP downstream effectors. This conclusion was based on the following observations: 1) the PKA antagonist H-89 completely prevented the effect of the PKA activator but only partially blocked the exendin-4-induced NHE3 inhibition; 2) the MEK1/2 inhibitor U-0126 abolished the effect of the EPAC activator but only diminished the exendin-4-induced NHE3 inhibition; 3) combination of H-89 and U-0126 fully prevented the effect of exendin-4 on NHE3; 4) no additive effect in the inhibition of NHE3 activity was observed when exendin-4, PKA, and EPAC activators were used together. Mechanistically, the inhibitory effect of exendin-4 on pHi recovery was associated with an increase of NHE3 phosphorylation. Conversely, this inhibition took place without changes in the surface expression of the transporter. We conclude that GLP-1 receptor agonists modulate sodium homeostasis in the kidney, most likely by affecting NHE3 activity.

Identification of Novel Interaction between ADAM17 (a Disintegrin and Metalloprotease 17) and Thioredoxin-1

ADAM17, which is also known as TNF alpha-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation.

Transforming growth factor β2 (TGF-β2)-induced connective tissue growth factor (CTGF) expression requires sphingosine 1-phosphate receptor 5 (S1P5) in human mesangial cells

Transforming growth factor β2 (TGF-β2) is well known to stimulate the expression of pro-fibrotic connective tissue growth factor (CTGF) in several cell types including human mesangial cells. The present study demonstrates that TGF-β2 enhances sphingosine 1-phosphate receptor 5 (S1P5) mRNA and protein expression in a time and concentration dependent manner. Pharmacological and siRNA approaches reveal that this upregulation is mediated via activation of classical TGF-β downstream effectors, Smad and mitogen-activated protein kinases. Most notably, inhibition of Gi with pertussis toxin and downregulation of S1P5 by siRNA block TGF-β2-stimulated upregulation of CTGF, demonstrating that Gi coupled S1P5 is necessary for TGF-β2-triggered expression of CTGF in human mesangial cells. Overall, these findings indicate that TGF-β2 dependent upregulation of S1P5 is required for the induction of pro-fibrotic CTGF by TGF-β. Targeting S1P5 might be an attractive novel approach to treat renal fibrotic diseases.

The chemokine monocyte chemoattractant protein-1 induces functional responses through dimerization of its receptor CCR2

Cytokines interact with hematopoietin superfamily receptors and stimulate receptor dimerization. We demonstrate that chemoattractant cytokines (chemokines) also trigger biological responses through receptor dimerization. Functional responses are induced after pairwise crosslinking of chemokine receptors by bivalent agonistic antichemokine receptor mAb, but not by their Fab fragments. Monocyte chemoattractant protein (MCP)-1-triggered receptor dimerization was studied in human embryonic kidney (HEK)-293 cells cotransfected with genes coding for the CCR2b receptor tagged with YSK or Myc sequences. After MCP-1 stimulation, immunoprecipitation with Myc-specific antibodies revealed YSK-tagged receptors in immunoblotting. Receptor dimerization also was validated by chemical crosslinking in both HEK-293 cells and the human monocytic cell line Mono Mac 1. Finally, we constructed a loss-of-function CCR2bY139F mutant that acted as a dominant negative, blocking signaling through the CCR2 wild-type receptor. This study provides functional support for a model in which the MCP-1 receptor is activated by ligand-induced homodimerization, allowing discussion of the similarities between bacterial and leukocyte chemotaxis.

T cell vaccination induces T cell receptor Vβ-specific Qa-1-restricted regulatory CD8+ T cells

Vaccination of mice with activated autoantigen-reactive CD4+ T cells (T cell vaccination, TCV) has been shown to induce protection from the subsequent induction of a variety of experimental autoimmune diseases, including experimental allergic encephalomyelitis (EAE). Although the mechanisms involved in TCV-mediated protection are not completely known, there is some evidence that TCV induces CD8+ regulatory T cells that are specific for pathogenic CD4+ T cells. Previously, we demonstrated that, after superantigen administration in vivo, CD8+ T cells emerge that preferentially lyse and regulate activated autologous CD4+ T cells in a T cell receptor (TCR) Vβ-specific manner. This TCR Vβ-specific regulation is not observed in β2-microglobulin-deficient mice and is inhibited, in vitro, by antibody to Qa-1. We now show that similar Vβ8-specific Qa-1-restricted CD8+ T cells are also induced by TCV with activated CD4+ Vβ8+ T cells. These CD8+ T cells specifically lyse murine or human transfectants coexpressing Qa-1 and murine TCR Vβ8. Further, CD8+ T cell hybridoma clones generated from B10.PL mice vaccinated with a myelin basic protein-specific CD4+Vβ8+ T cell clone specifically recognize other CD4+ T cells and T cell tumors that express Vβ8 and the syngeneic Qa-1a but not the allogeneic Qa-1b molecule. Thus, Vβ-specific Qa-1-restricted CD8+ T cells are induced by activated CD4+ T cells. We suggest that these CD8+ T cells may function to specifically regulate activated CD4+ T cells during immune responses.

Defects in inositol 1,4,5-trisphosphate receptor expression, Ca2+ signaling, and insulin secretion in the anx7(+/−) knockout mouse

The mammalian anx7 gene codes for a Ca2+-activated GTPase, which supports Ca2+/GTP-dependent secretion events and Ca2+ channel activities in vitro and in vivo. To test whether anx7 might be involved in Ca2+ signaling in secreting pancreatic β cells, we knocked out the anx7 gene in the mouse and tested the insulin-secretory properties of the β cells. The nullizygous anx7 (−/−) phenotype is lethal at embryonic day 10 because of cerebral hemorrhage. However, the heterozygous anx7 (+/−) mouse, although expressing only low levels of ANX7 protein, is viable and fertile. The anx7 (+/−) phenotype is associated with a substantial defect in insulin secretion, although the insulin content of the islets, is 8- to 10-fold higher in the mutants than in the normal littermate control. We infer from electrophysiological studies that both glucose-stimulated secretion and voltage-dependent Ca2+ channel functions are normal. However, electrooptical recordings indicate that the (+/−) mutation has caused a change in the ability of inositol 1,4,5-trisphosphate (IP3)-generating agonists to release intracellular calcium. The principle molecular consequence of lower anx7 expression is a profound reduction in IP3 receptor expression and function in pancreatic islets. The profound increase in islets, β cell number, and size may be a means of compensating for less efficient insulin secretion by individual defective pancreatic β cells. This is a direct demonstration of a connection between glucose-activated insulin secretion and Ca2+ signaling through IP3-sensitive Ca2+ stores.

Reciprocal interactions between β1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: A different perspective in epithelial biology

Anchorage and growth factor independence are cardinal features of the transformed phenotype. Although it is logical that the two pathways must be coregulated in normal tissues to maintain homeostasis, this has not been demonstrated directly. We showed previously that down-modulation of β1-integrin signaling reverted the malignant behavior of a human breast tumor cell line (T4–2) derived from phenotypically normal cells (HMT-3522) and led to growth arrest in a three-dimensional (3D) basement membrane assay in which the cells formed tissue-like acini (14). Here, we show that there is a bidirectional cross-modulation of β1-integrin and epidermal growth factor receptor (EGFR) signaling via the mitogen-activated protein kinase (MAPK) pathway. The reciprocal modulation does not occur in monolayer (2D) cultures. Antibody-mediated inhibition of either of these receptors in the tumor cells, or inhibition of MAPK kinase, induced a concomitant down-regulation of both receptors, followed by growth-arrest and restoration of normal breast tissue morphogenesis. Cross-modulation and tissue morphogenesis were associated with attenuation of EGF-induced transient MAPK activation. To specifically test EGFR and β1-integrin interdependency, EGFR was overexpressed in nonmalignant cells, leading to disruption of morphogenesis and a compensatory up-regulation of β1-integrin expression, again only in 3D. Our results indicate that when breast cells are spatially organized as a result of contact with basement membrane, the signaling pathways become coupled and bidirectional. They further explain why breast cells fail to differentiate in monolayer cultures in which these events are mostly uncoupled. Moreover, in a subset of tumor cells in which these pathways are misregulated but functional, the cells could be “normalized” by manipulating either pathway.

Activation of the orphan nuclear receptor steroidogenic factor 1 by oxysterols

Steroidogenic factor 1 (SF-1), an orphan member of the intracellular receptor superfamily, plays an essential role in the development and function of multiple endocrine organs. It is expressed in all steroidogenic tissues where it regulates the P450 steroidogenic genes to generate physiologically active steroids. Although many of the functions of SF-1 in vivo have been defined, an unresolved question is whether a ligand modulates its transcriptional activity. Here, we show that 25-, 26-, or 27-hydroxycholesterol, known suppressors of cholesterol biosynthesis, enhance SF-1-dependent transcriptional activity. This activation is dependent upon the SF-1 activation function domain, and, is specific for SF-1 as several other receptors do not respond to these molecules. The oxysterols activate at concentrations comparable to those previously shown to inhibit cholesterol biosynthesis, and, can be derived from cholesterol by P450c27, an enzyme expressed within steroidogenic tissues. Recent studies have shown that the nuclear receptor LXR also is activated by oxysterols. We demonstrate that different oxysterols differ in their rank order potency for these two receptors, with 25-hydroxycholesterol preferentially activating SF-1 and 22(R)-hydroxycholesterol preferentially activating LXR. These results suggest that specific oxysterols may mediate transcriptional activation via different intracellular receptors. Finally, ligand-dependent transactivation of SF-1 by oxysterols may play an important role in enhancing steroidogenesis in vivo.

A Cell Signal Pathway Involving Laminin-5, α3β1 Integrin, and Mitogen-activated Protein Kinase Can Regulate Epithelial Cell Proliferation

Laminin-5 (LN5) is a matrix component of epithelial tissue basement membranes and plays an important role in the initiation and maintenance of epithelial cell anchorage to the underlying connective tissue. Here we show that two distinct LN5 function-inhibitory antibodies, both of which bind the globular domain of the α3 subunit, inhibit proliferation of epithelial cells. These same antibodies also induce a decrease in mitogen-activated protein kinase activity. Inhibition of proliferation by the function-perturbing LN5 antibodies is reversed upon removal of the antibodies and can be overcome by providing the antibody-treated cells with exogenous LN5 and rat tail collagen. Because epithelial cells use the integrin receptor α3β1 to interact with both LN5 and rat tail collagen, we next investigated the possibility that integrin α3β1 is involved in mediating the proliferative impact of LN5. Proliferation of human epithelial cells is significantly inhibited by a function-perturbing α3 integrin antibody. In addition, antibody activation of β1 integrin restores the proliferation of epithelial cells treated with LN5 function-perturbing antibodies. These data indicate that a complex comprising LN5 and α3β1 integrin is multifunctional and contributes not only to epithelial cell adhesion but also to the regulation of cell growth via a signaling pathway involving mitogen-activated protein kinase. We discuss our study in light of recent evidence that LN5 expression is up-regulated at the leading tips of tumors, where it may play a role in tumor cell proliferation.

A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1

Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by the insulin receptor permits this docking protein to interact with signaling proteins that promote insulin action. Serine phosphorylation uncouples IRS-1 from the insulin receptor, thereby inhibiting its tyrosine phosphorylation and insulin signaling. For this reason, there is great interest in identifying serine/threonine kinases for which IRS-1 is a substrate. Tumor necrosis factor (TNF) inhibited insulin-promoted tyrosine phosphorylation of IRS-1 and activated the Akt/protein kinase B serine-threonine kinase, a downstream target for phosphatidylinositol 3-kinase (PI 3-kinase). The effect of TNF on insulin-promoted tyrosine phosphorylation of IRS-1 was blocked by inhibition of PI 3-kinase and the PTEN tumor suppessor, which dephosphorylates the lipids that mediate PI 3-kinase functions, whereas constitutively active Akt impaired insulin-promoted IRS-1 tyrosine phosphorylation. Conversely, TNF inhibition of IRS-1 tyrosine phosphorylation was blocked by kinase dead Akt. Inhibition of IRS-1 tyrosine phosphorylation by TNF was blocked by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a downstream target of Akt. mTOR induced the serine phosphorylation of IRS-1 (Ser-636/639), and such phosphorylation was inhibited by rapamycin. These results suggest that TNF impairs insulin signaling through IRS-1 by activation of a PI 3-kinase/Akt/mTOR pathway, which is antagonized by PTEN.

Mutant G protein α subunit activated by Gβγ: A model for receptor activation?

How receptors catalyze exchange of GTP for GDP bound to the Gα subunit of trimeric G proteins is not known. One proposal is that the receptor uses the G protein's βγ heterodimer as a lever, tilting it to pull open the guanine nucleotide binding pocket of Gα. To test this possibility, we designed a mutant Gα that would bind to βγ in the tilted conformation. To do so, we excised a helical turn (four residues) from the N-terminal region of αs, the α subunit of GS, the stimulatory regulator of adenylyl cyclase. In the presence, but not in the absence, of transiently expressed β1 and γ2, this mutant (αsΔ), markedly stimulated cAMP accumulation. This effect depended on the ability of the coexpressed β protein to interact normally with the lip of the nucleotide binding pocket of αsΔ. We substituted alanine for an aspartate in β1 that binds to a lysine (K206) in the lip of the α subunit's nucleotide binding pocket. Coexpressed with αsΔ and γ2, this mutant, β1-D228A, elevated cAMP much less than did β1-wild type; it did bind to αsΔ normally, however, as indicated by its unimpaired ability to target αsΔ to the plasma membrane. We conclude that βγ can activate αs and that this effect probably involves both a tilt of βγ relative to αs and interaction of β with the lip of the nucleotide binding pocket. We speculate that receptors use a similar mechanism to activate trimeric G proteins.

Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus.

The signaling pathways associated with estrogen-induced proliferation of epithelial cells in the reproductive tract have not been defined. To identify receptor tyrosine kinases that are activated in vivo by 17 beta-estradiol (E2), uteri from ovariectomized mice were examined for enhanced tyrosine phosphorylation of various receptors and a receptor substrate following treatment with this hormone. Within 4 hr after hormone exposure, extracts showed increased phosphotyrosine (P-Tyr) immunoreactivity at several bands, including 170- and 180-kDa; these bands were still apparent at 24 hr after E2. Analysis of immunoprecipitates from uterine extracts revealed that E2 enhanced tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor substrate-1 (IRS-1) by 6 hr. Comparison of supernatants from IRS-1 and control rabbit IgG immunoprecipitates indicated that the 170-kDa P-Tyr band in extracts was equivalent to IRS-1. The receptors for epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor did not exhibit an E2-induced increase in P-Tyr content. The nonestrogenic steroid hormones examined did not stimulate the P-Tyr content of IGF-1R or IRS-1. Immunolocalization of P-Tyr and IRS-1 revealed strong reactivity in the epithelial layer of the uterus from E2-treated mice, suggesting that the majority of P-Tyr bands observed in immunoblots originate in the epithelium. Since hormonal activation of IRS-1 is epithelial, estrogen-specific, and initiated before maximal DNA synthesis occurs following treatment with hormone, this protein, as part of the IGF-1R pathway, may be important in mediating estrogen-stimulated proliferation in the uterus.

The nuclear hormone receptor coactivator SRC-1 is a specific target of p300.

p300 and its family member, CREB-binding protein (CBP), function as key transcriptional coactivators by virtue of their interaction with the activated forms of certain transcription factors. In a search for additional cellular targets of p300/CBP, a protein-protein cloning strategy, surprisingly identified SRC-1, a coactivator involved in nuclear hormone receptor transcriptional activity, as a p300/CBP interactive protein. p300 and SRC-1 interact, specifically, in vitro and they also form complexes in vivo. Moreover, we show that SRC-1 encodes a new member of the basic helix-loop-helix-PAS domain family and that it physically interacts with the retinoic acid receptor in response to hormone binding. Together, these results implicate p300 as a component of the retinoic acid signaling pathway, operating, in part, through specific interaction with a nuclear hormone receptor coactivator, SRC-1.

Evidence for an insulin receptor substrate 1 independent insulin signaling pathway that mediates insulin-responsive glucose transporter (GLUT4) translocation.

Interaction of the activated insulin receptor (IR) with its substrate, insulin receptor substrate 1 (IRS-1), via the phosphotyrosine binding domain of IRS-1 and the NPXY motif centered at phosphotyrosine 960 of the IR, is important for IRS-1 phosphorylation. We investigated the role of this interaction in the insulin signaling pathway that stimulates glucose transport. Utilizing microinjection of competitive inhibitory reagents in 3T3-L1 adipocytes, we have found that disruption of the IR/IRS-1 interaction has no effect upon translocation of the insulin-responsive glucose transporter (GLUT4). The activity of these reagents was demonstrated by their ability to block insulin stimulation of two distinct insulin bioeffects, membrane ruffling and mitogenesis, in 3T3-L1 adipocytes and insulin-responsive rat 1 fibroblasts. These data suggest that phosphorylated IRS-1 is not an essential component of the metabolic insulin signaling pathway that leads to GLUT4 translocation, yet it appears to be required for other insulin bioeffects.