Pyrvinium Pamoate Use in a B cell Acute Lymphoblastic Leukemia Model of the Bone Tumor Microenvironment.

PURPOSE: Pyrvinium pamoate (PP) is an anthelmintic drug that has been found to have anti-cancer activity in several cancer types. In the present study, we evaluated PP for potential anti-leukemic activity in B cell acute lymphoblastic leukemia (ALL) cell lines, in an effort to evaluate the repurposing potential of this drug in leukemia. METHODS: ALL cells were treated with PP at various concentrations to determine its effect on cell proliferation. Metabolic function was tested by evaluating Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR). Lastly, 3D spheroids were grown, and PP was reformulated into nanoparticles to evaluate distribution effectiveness. RESULTS: PP was found to inhibit ALL proliferation, with varied selectivity to different ALL cell subtypes. We also found that PP's cell death activity was specific for leukemic cells, as primary normal immune cells were resistant to PP-mediated cell death. Metabolic studies indicated that PP, in part, inhibits mitochondrial oxidative phosphorylation. To increase the targeting of PP to a hypoxic bone tumor microenvironment (BTME) niche, we successfully encapsulated PP in a nanoparticle drug delivery system and demonstrated that it retained its anti-leukemic activity in a hemosphere assay. CONCLUSION: We have demonstrated that PP is a novel therapeutic lead compound that counteracts the respiratory reprogramming found in refractory ALL cells and can be effectively formulated into a nanoparticle delivery system to target the BTME.

The MitoNEET Ligand NL-1 Mediates Antileukemic Activity in Drug-Resistant B-Cell Acute Lymphoblastic Leukemia.

Disease relapse in B-cell acute lymphoblastic leukemia (ALL), either due to development of acquired resistance after therapy or because of de novo resistance, remains a therapeutic challenge. In the present study, we have developed a cytarabine (Ara-C)-resistant REH cell line (REH/Ara-C) as a chemoresistance model. REH/Ara-C 1) was not crossresistant to vincristine or methotrexate; 2) showed a similar proliferation rate and cell surface marker expression as parental REH; 3) demonstrated decreased chemotaxis toward bone marrow stromal cells; and 4) expressed higher transcript levels of cytidine deaminase (CDA) and mitoNEET (CISD1) than the parental REH cell line. Based on these findings, we tested NL-1, a mitoNEET inhibitor, which induced a concentration-dependent decrease in cell viability with a comparable IC50 value in REH and REH/Ara-C. Furthermore, NL-1 decreased cell viability in six different ALL cell lines and showed inhibitory activity in a hemosphere assay. NL-1 also impaired the migratory ability of leukemic cells, irrespective of the chemoattractant used, in a chemotaxis assay. More importantly, NL-1 showed specific activity in inducing death in a drug-resistant population of leukemic cells within a coculture model that mimicked the acquired resistance and de novo resistance observed in the bone marrow of relapsed patients. Subsequent studies indicated that NL-1 mediates autophagy, and inhibition of autophagy partially decreased NL-1-induced tumor cell death. Finally, NL-1 showed antileukemic activity in an in vivo mouse ALL model. Taken together, our study demonstrates that mitoNEET has potential as a novel antileukemic drug target in treatment refractory or relapsed ALL.

SOX9 inhibits ß-TrCP-mediated protein degradation to promote nuclear GLI1 expression and cancer stem cell properties.

The high mobility group box protein SOX9 and the GLI1 transcription factor play protumorigenic roles in pancreatic ductal adenocarcinoma (PDA). In Kras transgenic mice, each of these factors are crucial for the development of PDA precursor lesions. SOX9 transcription is directly regulated by GLI1, but how SOX9 functions downstream of GLI1 is unclear. We observed positive feedback, such that SOX9-deficient PDA cells have severely repressed levels of endogenous GLI1, attributed to loss of GLI1 protein stability. SOX9 associated with the F-box domain of the SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase component, ß-TrCP (also known as F-box/WD repeat-containing protein 1A), and suppressed its association with SKP1 and GLI1, a substrate of SCF-ß-TrCP. SOX9 also tethered ß-TrCP within the nucleus and promoted its degradation. SOX9 bound to ß-TrCP through the SOX9 C-terminal PQA/S domain that mediates transcriptional activation. Suppression of ß-TrCP in SOX9-deficient PDA cells restored GLI1 levels and promoted SOX9-dependent cancer stem cell properties. These studies identify SOX9-GLI1 positive feedback as a major determinant of GLI1 protein stability and implicate ß-TrCP as a latent SOX9-bound tumor suppressor with the potential to degrade oncogenic proteins in tumor cells.

Leptin decreases heart rate associated with increased ventricular repolarization via its receptor.

Leptin has been proposed to modulate cardiac electrical properties via ß-adrenergic receptor activation. The presence of leptin receptors and adipocytes in myocardium raised a question as to whether leptin can directly modulate cardiac electrical properties such as heart rate and QT interval via its receptor. In this work, the role of local direct actions of leptin on heart rate and ventricular repolarization was investigated. We identified the protein expression of leptin receptors at cell surface of sinus node, atrial, and ventricular myocytes isolated from rat heart. Leptin at low doses (0.1-30 µg/kg) decreased resting heart rate; at high doses (150-300 µg/kg), leptin induced a biphasic effect (decrease and then increase) on heart rate. In the presence of high-dose propranolol (30 mg/kg), high-dose leptin only reduced heart rate and sometimes caused sinus pauses and ventricular tachycardia. The leptin-induced inhibition of resting heart rate was fully reversed by leptin antagonist. Leptin also increased heart rate-corrected QT interval (QTc), and leptin antagonist did not. In isolated ventricular myocytes, leptin (0.03-0.3 µg/ml) reversibly increased the action potential duration. These results supported our hypothesis that in addition to indirect pathway via sympathetic tone, leptin can directly decrease heart rate and increase QT interval via its receptor independent of ß-adrenergic receptor stimulation. During inhibition of ß-adrenergic receptor activity, high concentration of leptin in myocardium can cause deep bradycardia, prolonged QT interval, and ventricular arrhythmias.

PP2 prevents isoproterenol stimulation of cardiac pacemaker activity.

Increasing evidence has demonstrated the potential risks of cardiac arrhythmias (such as prolonged QT interval) using tyrosine kinase inhibitors for cancer therapy. We report here that a widely used selective inhibitor of Src tyrosine kinases, PP2, can inhibit and prevent isoproterenol stimulation of cardiac pacemaker activity. In dissected rat sinus node, PP2 inhibited and prevented isoproterenol stimulation of spontaneous beating rate. In isolated sinus node myocytes, PP2 suppressed the hyperpolarization-activated "funny" current (If) by negatively shifting the activation curve and decelerating activation kinetics, associated with decreased cell surface expression and reduced tyrosine phosphorylation of hyperpolarization-activated cyclic nucleotide-modulated channel 4 (HCN4) channel proteins. In human embryonic kidney 293 cells overexpressing recombinant human HCN4 channels, PP2 reversed isoproterenol stimulation of HCN4 and inhibited HCN4-573x, a cAMP-insensitive human HCN4 mutant. Isoprotenrenol had little effects on HCN4-573x. These results demonstrated that inhibition of presumably tyrosine Src kinase activity in heart by PP2 decreased and prevented the potential ß-adrenergic stimulation of cardiac pacemaker activity. These effects are mediated, at least partially, by a cAMP-independent attenuation of channel activity and cell surface expression of HCN4, the key channel protein that controls the heart rate.

Induction of stemlike cells with fibrogenic properties by carbon nanotubes and its role in fibrogenesis.

We developed a three-dimensional fibroblastic nodule model for fibrogenicity testing of nanomaterials and investigated the role of fibroblast stemlike cells (FSCs) in the fibrogenic process. We showed that carbon nanotubes (CNTs) induced fibroblastic nodule formation in primary human lung fibroblast cultures resembling the fibroblastic foci in clinical fibrosis and promoted FSCs that are highly fibrogenic and a potential driving force of fibrogenesis. This study provides a predictive 3D model and mechanistic insight on CNT fibrogenesis.

The group A streptococcal collagen-like protein-1, Scl1, mediates biofilm formation by targeting the extra domain A-containing variant of cellular fibronectin expressed in wounded tissue.

Wounds are known to serve as portals of entry for group A Streptococcus (GAS). Subsequent tissue colonization is mediated by interactions between GAS surface proteins and host extracellular matrix components. We recently reported that the streptococcal collagen-like protein-1, Scl1, selectively binds the cellular form of fibronectin (cFn) and also contributes to GAS biofilm formation on abiotic surfaces. One structural feature of cFn, which is predominantly expressed in response to tissue injury, is the presence of a spliced variant containing extra domain A (EDA/EIIIA). We now report that GAS biofilm formation is mediated by the Scl1 interaction with EDA-containing cFn. Recombinant Scl1 proteins that bound cFn also bound recombinant EDA within the C-C' loop region recognized by the α(9)ß(1) integrin. The extracellular 2-D matrix derived from human dermal fibroblasts supports GAS adherence and biofilm formation. Altogether, this work identifies and characterizes a novel molecular mechanism by which GAS utilizes Scl1 to specifically target an extracellular matrix component that is predominantly expressed at the site of injury in order to secure host tissue colonization.

Src binds cortactin through an SH2 domain cystine-mediated linkage.

Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.

PP2 prevents ß-adrenergic stimulation of cardiac pacemaker activity.

One of the main strategies for cancer therapy is to use tyrosine kinase inhibitors for inhibiting tumor proliferation. Increasing evidence has demonstrated the potential risks of cardiac arrhythmias (such as prolonged QT interval) of these drugs. We report here that a widely used selective inhibitor of Src tyrosine kinases, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), can inhibit and prevent ß-adrenergic stimulation of cardiac pacemaker activity. First, in dissected rat sinus node, PP2 inhibited and prevented the isoproterenol-induced increase of spontaneous beating rate. Second, in isolated rat sinus node myocytes, PP2 suppressed the hyperpolarization-activated "funny" current (traditionally called cardiac pacemaker current, I(f)) by negatively shifting the activation curve and decelerating activation kinetics. Third, in isolated rat sinus node myocytes, PP2 decreased the Src kinase activity, the cell surface expression, and tyrosine phosphorylation of hyperpolarization-activated, cyclic nucleotide-modulated channel 4 (HCN4) channel proteins. Finally, in human embryonic kidney 293 cells overexpressing recombinant human HCN4 channels, PP2 reversed the enhancement of HCN4 channels by isoproterenol and inhibited 573x, a cyclic adenosine momophosphate-insensitive human HCN4 mutant. These results demonstrated that inhibition of Src kinase activity in heart by PP2 decreased and prevented ß-adrenergic stimulation of cardiac pacemaker activity. These effects are mediated, at least partially, by a cAMP-independent attenuation of channel activity and cell surface expression of HCN4, the main channel protein that controls the heart rate.

Chemotherapeutic Activity of Pitavastatin in Vincristine Resistant B-Cell Acute Lymphoblastic Leukemia.

B-cell acute lymphoblastic leukemia (ALL) is derived from an accumulation of malignant, immature B cells in the bone marrow and blood. Relapse due, in part, to the emergence of tumor cells that are resistant to front line standard chemotherapy is associated with poor patient outcomes. This challenge highlights the need for new treatment strategies to eliminate residual chemoresistant tumor cells. Based on the use of pitavastatin in acute myeloid leukemia (AML), we evaluated its efficacy in an REH ALL cell line derived to be resistant to vincristine. We found that pitavastatin inhibited the proliferation of both parental and vincristine-resistant REH tumor cells at an IC50 of 449 nM and 217 nM, respectively. Mitochondrial bioenergetic assays demonstrated that neither vincristine resistance nor pitavastatin treatment affected cellular oxidative phosphorylation, beta-oxidation, or glycolytic metabolism in ALL cells. In a co-culture model of ALL cells with bone marrow stromal cells, pitavastatin significantly decreased cell viability more robustly in the vincristine-resistant ALL cells compared with their parental controls. Subsequently, NSG mice were used to develop an in vivo model of B-cell ALL using both parental and vincristine-resistant ALL cells. Pitavastatin (10 mg/kg i.p.) significantly reduced the number of human CD45+ REH ALL cells in the bone marrow of mice after 4 weeks of treatment. Mechanistic studies showed that pitavastatin treatment in the vincristine-resistant cells led to apoptosis, with increased levels of cleaved PARP and protein-signaling changes for AMP-activated protein kinase/FoxO3a/Puma. Our data suggest the possible repurposing of pitavastatin as a chemotherapeutic agent in a model of vincristine-resistant B-cell ALL.

Examining the role of actin-plasma membrane association in Pseudomonas aeruginosa infection and type III secretion translocation in migratory T24 epithelial cells.

The opportunistic pathogen Pseudomonas aeruginosa targets wounded epithelial barriers, but the cellular alteration that increases susceptibility to P. aeruginosa infection remains unclear. This study examined how cell migration contributes to the establishment of P. aeruginosa infections using (i) highly migratory T24 epithelial cells as a cell culture model, (ii) mutations in the type III secretion (T3S) effector ExoS to manipulate P. aeruginosa infection, and (iii) high-resolution immunofluorescent microscopy to monitor ExoS translocation. ExoS includes both GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) activities, and P. aeruginosa cells expressing wild-type ExoS preferentially bound to the leading edge of T24 cells, where ExoS altered leading-edge architecture and actin anchoring in conjunction with interrupting T3S translocation. Inactivation of ExoS GAP activity allowed P. aeruginosa to be internalized and secrete ExoS within T24 cells, but as with wild-type ExoS, translocation was limited in association with disruption of actin anchoring. Inactivation of ExoS ADPRT activity resulted in significantly enhanced T3S translocation by P. aeruginosa cells that remained extracellular and in conjunction with maintenance of actin-plasma membrane association. Infection with P. aeruginosa expressing ExoS lacking both GAP and ADPRT activities resulted in the highest level of T3S translocation, and this occurred in conjunction with the entry and alignment of P. aeruginosa and ExoS along actin filaments. Collectively, in using ExoS mutants to modulate and visualize T3S translocation, we were able to (i) confirm effector secretion by internalized P. aeruginosa, (ii) differentiate the mechanisms underlying the effects of ExoS GAP and ADPRT activities on P. aeruginosa internalization and T3S translocation, (iii) confirm that ExoS ADPRT activity targeted a cellular substrate that interrupted T3S translocation, (iv) visualize the ability of P. aeruginosa and ExoS to align with actin filaments, and (v) demonstrate an association between actin anchoring at the leading edge of T24 cells and the establishment of P. aeruginosa infection. Our studies also highlight the contribution of ExoS to the opportunistic nature of P. aeruginosa infection through its ability to exert cytotoxic effects that interrupt T3S translocation and P. aeruginosa internalization, which in turn limit the P. aeruginosa infectious process.

Oncogenic Src requires a wild-type counterpart to regulate invadopodia maturation.

The proto-oncogene Src tyrosine kinase (Src) is overexpressed in human cancers and is currently a target of anti-invasive therapies. Activation of Src is an essential catalyst of invadopodia production. Invadopodia are cellular structures that mediate extracellular matrix (ECM) proteolysis, allowing invasive cell types to breach confining tissue barriers. Invadopodia assembly and maturation is a multistep process, first requiring the targeting of actin-associated proteins to form pre-invadopodia, which subsequently mature by recruitment and activation of matrix metalloproteases (MMPs) that facilitate ECM degradation. We demonstrate that active, oncogenic Src alleles require the presence of a wild-type counterpart to induce ECM degradation at invadopodia sites. In addition, we identify the phosphorylation of the invadopodia regulatory protein cortactin as an important mediator of invadopodia maturation downstream of wild-type Src. Distinct phosphotyrosine-based protein-binding profiles in cells forming pre-invadopodia and mature invadopodia were identified by SH2-domain array analysis. These results indicate that although elevated Src kinase activity is required to target actin-associated proteins to pre-invadopodia, regulated Src activity is required for invadopodia maturation and matrix degradation activity. Our findings describe a previously unappreciated role for proto-oncogenic Src in enabling the invasive activity of constitutively active Src alleles.

Pitavastatin Is Anti-Leukemic in a Bone Marrow Microenvironment Model of B-Lineage Acute Lymphoblastic Leukemia.

The lack of complete therapeutic success in the treatment of B-cell acute lymphoblastic leukemia (ALL) has been attributed, in part, to a subset of cells within the bone marrow microenvironment that are drug resistant. Recently, the cholesterol synthesis inhibitor, pitavastatin (PIT), was shown to be active in acute myeloid leukemia, prompting us to evaluate it in our in vitro co-culture model, which supports a chemo-resistant ALL population. We used phospho-protein profiling to evaluate the use of lipid metabolic active compounds in these chemo-resistant cells, due to the up-regulation of multiple active survival signals. In a co-culture with stromal cells, a shift towards anabolic processes occurred, which was further confirmed by assays showing increased lipid content. The treatment of REH leukemia cells with pitavastatin in the co-culture model resulted in significantly higher leukemic cell death than exposure to the standard-of-care chemotherapeutic agent, cytarabine (Ara-C). Our data demonstrates the use of pitavastatin as a possible alternative treatment strategy to improve patient outcomes in chemo-resistant, relapsed ALL.

The role of calcium release activated calcium channels in osteoclast differentiation.

Osteoclasts are specialized macrophage derivatives that secrete acid and proteinases to mobilize bone for mineral homeostasis, growth, and replacement or repair. Osteoclast differentiation generally requires the monocyte growth factor m-CSF and the TNF-family cytokine RANKL, although differentiation is regulated by many other cytokines and by intracellular signals, including Ca(2+). Studies of osteoclast differentiation in vitro were performed using human monocytic precursors stimulated with m-CSF and RANKL, revealing significant loss in both the expression and function of the required components of store-operated Ca(2+) entry over the course of osteoclast differentiation. However, inhibition of CRAC using either the pharmacological agent 3,4-dichloropropioanilide (DCPA) or by knockdown of Orai1 severely inhibited formation of multinucleated osteoclasts. In contrast, no effect of CRAC channel inhibition was observed on expression of the osteoclast protein tartrate resistant acid phosphatase (TRAP). Our findings suggest that despite the fact that they are down-regulated during osteoclast differentiation, CRAC channels are required for cell fusion, a late event in osteoclast differentiation. Since osteoclasts cannot function properly without multinucleation, selective CRAC inhibitors may have utility in management of hyperresorptive states.

The streptococcal collagen-like protein-1 (Scl1) is a significant determinant for biofilm formation by group A Streptococcus.

BACKGROUND: Group A Streptococcus (GAS) is a human-specific pathogen responsible for a number of diseases characterized by a wide range of clinical manifestations. During host colonization GAS-cell aggregates or microcolonies are observed in tissues. GAS biofilm, which is an in vitro equivalent of tissue microcolony, has only recently been studied and little is known about the specific surface determinants that aid biofilm formation. In this study, we demonstrate that surface-associated streptococcal collagen-like protein-1 (Scl1) plays an important role in GAS biofilm formation. RESULTS: Biofilm formation by M1-, M3-, M28-, and M41-type GAS strains, representing an intraspecies breadth, were analyzed spectrophotometrically following crystal violet staining, and characterized using confocal and field emission scanning electron microscopy. The M41-type strain formed the most robust biofilm under static conditions, followed by M28- and M1-type strains, while the M3-type strains analyzed here did not form biofilm under the same experimental conditions. Differences in architecture and cell-surface morphology were observed in biofilms formed by the M1- and M41-wild-type strains, accompanied by varying amounts of deposited extracellular matrix and differences in cell-to-cell junctions within each biofilm. Importantly, all Scl1-negative mutants examined showed significantly decreased ability to form biofilm in vitro. Furthermore, the Scl1 protein expressed on the surface of a heterologous host, Lactococcus lactis, was sufficient to induce biofilm formation by this organism. CONCLUSIONS: Overall, this work (i) identifies variations in biofilm formation capacity among pathogenically different GAS strains, (ii) identifies GAS surface properties that may aid in biofilm stability and, (iii) establishes that the Scl1 surface protein is an important determinant of GAS biofilm, which is sufficient to enable biofilm formation in the heterologous host Lactococcus. In summary, the GAS surface adhesin Scl1 may have an important role in biofilm-associated pathogenicity.

Co-culture model of B-cell acute lymphoblastic leukemia recapitulates a transcription signature of chemotherapy-refractory minimal residual disease.

B-cell acute lymphoblastic leukemia (ALL) is characterized by accumulation of immature hematopoietic cells in the bone marrow, a well-established sanctuary site for leukemic cell survival during treatment. While standard of care treatment results in remission in most patients, a small population of patients will relapse, due to the presence of minimal residual disease (MRD) consisting of dormant, chemotherapy-resistant tumor cells. To interrogate this clinically relevant population of treatment refractory cells, we developed an in vitro cell model in which human ALL cells are grown in co-culture with human derived bone marrow stromal cells or osteoblasts. Within this co-culture, tumor cells are found in suspension, lightly attached to the top of the adherent cells, or buried under the adherent cells in a population that is phase dim (PD) by light microscopy. PD cells are dormant and chemotherapy-resistant, consistent with the population of cells that underlies MRD. In the current study, we characterized the transcriptional signature of PD cells by RNA-Seq, and these data were compared to a published expression data set derived from human MRD B-cell ALL patients. Our comparative analyses revealed that the PD cell population is markedly similar to the MRD expression patterns from the primary cells isolated from patients. We further identified genes and key signaling pathways that are common between the PD tumor cells from co-culture and patient derived MRD cells as potential therapeutic targets for future studies.

Loss of the redox mitochondrial protein mitoNEET leads to mitochondrial dysfunction in B-cell acute lymphoblastic leukemia.

B-cell acute lymphoblastic leukemia (ALL) affects both pediatric and adult patients. Chemotherapy resistant tumor cells that contribute to minimal residual disease (MRD) underlie relapse and poor clinical outcomes in a sub-set of patients. Targeting mitochondrial oxidative phosphorylation (OXPHOS) in the treatment of refractory leukemic cells is a potential novel approach to sensitizing tumor cells to existing standard of care therapeutic agents. In the current study, we have expanded our previous investigation of the mitoNEET ligand NL-1 in the treatment of ALL to interrogate the functional role of the mitochondrial outer membrane protein mitoNEET in B-cell ALL. Knockout (KO) of mitoNEET (gene: CISD1) in REH leukemic cells led to changes in mitochondrial ultra-structure and function. REH cells have significantly reduced OXPHOS capacity in the KO cells coincident with reduction in electron flow and increased reactive oxygen species. In addition, we found a decrease in lipid content in KO cells, as compared to the vector control cells was observed. Lastly, the KO of mitoNEET was associated with decreased proliferation as compared to control cells when exposed to the standard of care agent cytarabine (Ara-C). Taken together, these observations suggest that mitoNEET is essential for optimal function of mitochondria in B-cell ALL and may represent a novel anti-leukemic drug target for treatment of minimal residual disease.

Inactivation of L-type calcium channel modulated by HCN2 channel.

Ca(2+) entry is delicately controlled by inactivation of L-type calcium channel (LTCC) composed of the pore-forming subunit alpha1C and the auxiliary subunits beta1 and alpha2delta. Calmodulin is the key protein that interacts with the COOH-terminal motifs of alpha1C, leading to the fine control of LTCC inactivation. In this study we show evidence that a hyperpolarization-activated cyclic nucleotide-gated channel, HCN2, can act as a nonchannel regulatory protein to narrow the L-type Ca(2+) channel current-voltage curve. In the absence of LTCC auxiliary subunits, HCN2 can induce alpha1C inactivation. Without alpha2delta, HCN2-induced fast inactivation of alpha1C requires calmodulin. With alpha2delta, the alpha1C/HCN2/alpha2delta channel inactivation does not require calmodulin. In contrast, beta1-subunit plays a relatively minor role in the interaction of alpha1C with HCN2. The NH(2) terminus of HCN2 and the IQ motif of alpha1C subunit are required for alpha1C/HCN2 channel interaction. Ca(2+) channel inactivation is significantly slowed in hippocampus neurons (HNs) overexpressing HCN2 mutant lacking NH(2) terminus and accelerated in HNs overexpressing the wild-type HCN2 compared with HN controls. Collectively, these results revealed a potentially novel protection mechanism for achieving the LTCC inactivation via interaction with HCN2.

A(1) adenosine receptor-mediated PKC and p42/p44 MAPK signaling in mouse coronary artery smooth muscle cells.

The A(1) adenosine receptor (A(1)AR) is coupled to G(i)/G(o) proteins, but the downstream signaling pathways in smooth muscle cells are unclear. This study was performed in coronary artery smooth muscle cells (CASMCs) isolated from the mouse heart [A(1)AR wild type (A(1)WT) and A(1)AR knockout (A(1)KO)] to delineate A(1)AR signaling through the PKC pathway. In A(1)WT cells, treatment with (2S)-N(6)-(2-endo-norbornyl)adenosine (ENBA; 10(-5)M) increased A(1)AR expression by 150%, which was inhibited significantly by the A(1)AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (10(-6)M), but not in A(1)KO CASMCs. PKC isoforms were identified by Western blot analysis in the cytosolic and membrane fractions of cell homogenates of CASMCs. In A(1)WT and A(1)KO cells, significant levels of basal PKC-alpha were detected in the cytosolic fraction. Treatment with the A(1)AR agonist ENBA (10(-5)M) translocated PKC-alpha from the cytosolic to membrane fraction significantly in A(1)WT but not A(1)KO cells. Phospholipase C isoforms (betaI, betaIII, and gamma(1)) were analyzed using specific antibodies where ENBA treatment led to the increased expression of PLC-betaIII in A(1)WT CASMCs while having no effect in A(1)KO CASMCs. In A(1)WT cells, ENBA increased PKC-alpha expression and p42/p44 MAPK (ERK1/2) phospohorylation by 135% and 145%, respectively. These effects of ENBA were blocked by Gö-6976 (PKC-alpha inhibitor) and PD-98059 (p42/p44 MAPK inhibitor). We conclude that A(1)AR stimulation by ENBA activates the PKC-alpha signaling pathway, leading to p42/p44 MAPK phosphorylation in CASMCs.

The association of ASAP1, an ADP ribosylation factor-GTPase activating protein, with focal adhesion kinase contributes to the process of focal adhesion assembly.

ASAP1 (ADP ribosylation factor [ARF]- GTPase-activating protein [GAP] containing SH3, ANK repeats, and PH domain) is a phospholipid-dependent ARF-GAP that binds to and is phosphorylated by pp60(Src). Using affinity chromatography and yeast two-hybrid interaction screens, we identified ASAP1 as a major binding partner of protein tyrosine kinase focal adhesion kinase (FAK). Glutathione S-transferase pull-down and coimmunoprecipitation assays showed the binding of ASAP1 to FAK is mediated by an interaction between the C-terminal SH3 domain of ASAP1 with the second proline-rich motif in the C-terminal region of FAK. Transient overexpression of wild-type ASAP1 significantly retarded the spreading of REF52 cells plated on fibronectin. In contrast, overexpression of a truncated variant of ASAP1 that failed to bind FAK or a catalytically inactive variant of ASAP1 lacking GAP activity resulted in a less pronounced inhibition of cell spreading. Transient overexpression of wild-type ASAP1 prevented the efficient organization of paxillin and FAK in focal adhesions during cell spreading, while failing to significantly alter vinculin localization and organization. We conclude from these studies that modulation of ARF activity by ASAP1 is important for the regulation of focal adhesion assembly and/or organization by influencing the mechanisms responsible for the recruitment and organization of selected focal adhesion proteins such as paxillin and FAK.