Induce Actin Polymerization Research Articles

Molecular Basis of LH Action on Breast Cancer Cell Migration and Invasion via Kinase and Scaffold Proteins.

Breast cancer (BC) is a major public health problem affecting women worldwide. Approximately 80% of diagnosed cases are hormone-dependent breast cancers. These hormones are known to stimulate tumor development and progression. In this setting, tentative evidence suggests that luteinizing hormone (LH) may also play a role in tumors. In BC cells that express functional LH receptors (LHR), this hormone regulates cell migration and invasion by controlling several kinases that activate actin cytoskeletal proteins. In this article, we show that LH induces phosphorylation of paxillin and its translocation toward the plasmatic membrane, where focal adhesion complexes are assembled. This process is triggered via a rapid extra-gonadal LHR signaling to Src/FAK/paxillin, which results in the phosphorylation/activation of the nucleation promoter factors cortactin and N-WASP. As a consequence, Arp2/3 complexes induce actin polymerization, essential to promote cell adhesion, migration, and invasion, thus enhancing metastatic spread of tumoral cells. Our findings provide relevant information about how gonadotrophins exert their action in BC. This information helps us understand the extragonadal effects of LH on BC metastasis. It may provide new perspectives for therapeutic treatment, especially for women with high serum levels of gonadotrophins.

Local Myo9b RhoGAP activity regulates cell motility

To migrate, cells assume a polarized morphology, extending forward with a leading edge with their trailing edge retracting back toward the cell body. Both cell extension and retraction critically depend on the organization and dynamics of the actin cytoskeleton, and the small, monomeric GTPases Rac and Rho are important regulators of actin. Activation of Rac induces actin polymerization and cell extension, whereas activation of Rho enhances acto-myosin II contractility and cell retraction. To coordinate migration, these processes must be carefully regulated. The myosin Myo9b, a Rho GTPase-activating protein (GAP), negatively regulates Rho activity and deletion of Myo9b in leukocytes impairs cell migration through increased Rho activity. However, it is not known whether cell motility is regulated by global or local inhibition of Rho activity by Myo9b. Here, we addressed this question by using Myo9b-deficient macrophage-like cells that expressed different recombinant Myo9b constructs. We found that Myo9b accumulates in lamellipodial extensions generated by Rac-induced actin polymerization as a function of its motor activity. Deletion of Myo9b in HL-60–derived macrophages altered cell morphology and impaired cell migration. Reintroduction of Myo9b or Myo9b motor and GAP mutants revealed that local GAP activity rescues cell morphology and migration. In summary, Rac activation leads to actin polymerization and recruitment of Myo9b, which locally inhibits Rho activity to enhance directional cell migration.

Pharmacological Targeting Of Transgelin-2 As A Novel Strategy Of Therapeutic Intervention In Glioblastoma

GBM patients with wild-type IDH1/2 experience worse survival response to the standard treatment comprising surgery followed by radiation and temozolomide chemotherapy, compared with those harboring mutant IDH1/2. The treatment has remained relatively ineffective partly due to their invasive phenotype leading to tumor recurrence. In our clinical correlative studies, Transgelin-2 was found to be one of the top candidate genes associated with worse patient outcome. Transgelin-2 is known to induce actin polymerization, thereby playing a critical role in cytoskeletal reorganization leading to cell proliferation, migration/invasion, and therapeutic resistance in GBM and other malignancies. This study aims to establish Transgelin-2 as a potential therapeutic target in GBM and to demonstrate that targeting transgelin-2 by pharmacological inhibitors would provide a novel strategy for therapeutic intervention. RNAi-mediated TAGLN-2 knockdown (KD) approach was employed to assess the functions of Transgelin-2 in PDX GBM cell lines. A series of in-vitro functional assays was performed to assess the role of Transgelin-2 in these cell lines. Structure-based computational screening approach was employed to identify small-molecule inhibitors of Transgelin-2. Subsequently, the efficacy of these inhibitors to inhibit Transgelin-2-mediated cell proliferation/survival, invasion, and colony-formation was evaluated. RNAi-mediated silencing Transgelin-2 significantly inhibited proliferation, survival, clonal expansion, and invasion of GBM 08-387 and 3359 PDX cell lines in vitro. In-silico virtual screenings identified potential Transgelin-2 binding candidates and were predicted to be able to permeate blood-brain barrier. In vitro binding study confirmed that these compounds inhibit the Transgelin-2-dependent actin-polymerization, and significantly compromised the proliferation invasion of GBM cells as assessed by proliferation, survival, clonal expansion, and invasion of GBM 08-387 and 3359 PDX cell lines. Furthermore, it is noteworthy that it is more likely that other pro-invasion pathways like MMP2/9-pathways may compensate for loss of function of transgelin-2 and needs to be addressed as well. MMP2 and MMP9 are important determinants in cell migration/invasion and found to be differentially upregulated in human GBM tissue compared to normal brain tissue Therefore, a combinatorial approach including Transgelin-2 inhibitor and MMP inhibitor may provide additive/synergistic outcome. Our data suggest that Transgelin-2 may serve as a potential therapeutically vulnerable target in GBM. Importantly, select small molecule inhibitors of Transgelin-2 that inhibit cell proliferation, invasion and therapeutic resistance may have potential preclinical trials.

Fibrinogen/AKT/Microfilament Axis Promotes Colitis by Enhancing Vascular Permeability

Background & AimsIncreased vascular permeability (VP) has been indicated to play an important role in the pathogenesis of inflammatory bowel disease (IBD). However, the pathological causes of increased intestinal VP in IBD remain largely unknown.MethodFibrinogen level was measured in dextran sulphate sodium (DSS)-induced colitis and patients with ulcerative colitis. Gly-Pro-Arg-Pro acetate (GPRP), an Fg inhibitor, was used to detect the effect of Fg inhibition on the pathogenesis of DSS-induced colitis, as indicated by tissue damage, cytokine release and inflammatory cell infiltration. Miles assay was used to detect vascular permeability.ResultsThrough tandem mass tag–based quantitative proteomics, fibrinogen (Fg) was found to be upregulated in the colon of DSS-treated mice, which was consistent with increased Fg level in colon sample of patients with ulcerative colitis. Gly-Pro-Arg-Pro acetate (GPRP), an Fg inhibitor, significantly alleviated DSS-induced colitis as indicated by improvement of body weight loss and mortality. GPRP decreased colonic inflammation and VP in DSS-treated mice. In vivo, Fg enhanced VP as indicated by Miles assay, which was significantly inhibited by GRPR, AKT (serine/threonine kinase 1) inhibitors and low doses of Jasplakinolide which induced actin polymerization, while was dramatically enhanced by Cytochalasin D (an actin polymerization inhibitor). Moreover, activation of AKT was found in vessels of DSS-treated mice. In vitro, Fg induced activation of AKT and depolymerization of microfilament and promoted cell-to-cell disaggregation. Furthermore, inhibition of AKT decreased Fg-induced microfilament depolymerization.ConclusionsOur findings highlight the importance of Fg in regulating colitis by modulation of VP via activating AKT and subsequent depolymerization of microfilament and suggest Fg as an attractive target for anti-colitis treatment.

Ste20-like Kinase-mediated Control of Actin Polymerization Is a New Mechanism for Thin Filament-associated Regulation of Airway Smooth Muscle Contraction.

It has been reported that actin polymerization is regulated by protein tyrosine phosphorylation in smooth muscle on contractile stimulation. The role of protein serine/threonine phosphorylation in modulating actin dynamics is underinvestigated. SLK (Ste20-like kinase) is a serine/threonine protein kinase that plays a role in apoptosis, cell cycle, proliferation, and migration. The function of SLK in smooth muscle is mostly unknown. Here, SLK knockdown (KD) inhibited acetylcholine (ACh)-induced actin polymerization and contraction without affecting myosin light chain phosphorylation at Ser-19 in human airway smooth muscle. Stimulation with ACh induced paxillin phosphorylation at Ser-272, which was reduced in SLK KD cells. However, SLK did not catalyze paxillin Ser-272 phosphorylation in vitro. But, SLK KD attenuated Plk1 (polo-like kinase 1) phosphorylation at Thr-210. Plk1 mediated paxillin phosphorylation at Ser-272 in vitro. Expression of the nonphosphorylatable paxillin mutant S272A (substitution of alanine at Ser-272) attenuated the agonist-enhanced F-actin/G-actin ratios without affecting myosin light chain phosphorylation. Because N-WASP (neuronal Wiskott-Aldrich Syndrome Protein) phosphorylation at Tyr-256 (an indication of its activation) promotes actin polymerization, we also assessed the role of paxillin phosphorylation in N-WASP activation. S272A paxillin inhibited the ACh-enhanced N-WASP phosphorylation at Tyr-256. Together, these results suggest that SLK regulates paxillin phosphorylation at Ser-272 via Plk1, which modulates N-WASP activation and actin polymerization in smooth muscle. SLK-mediated actin cytoskeletal reorganization may facilitate force transmission between the contractile units and the extracellular matrix.

Wnt5A Signaling Mediated Cytoskeletal Actin Modulations Shape the Outcome of Host‐Bacteria Interactions

Many human diseases are caused by bacterial infections. Host factors play an important role in determining the persistence vs. eradication of the incumbent bacteria. Among the milieu of host factors involved in immune defense against infections, Wnt5A‐Frizzled5/ROR1 signaling has emerged as an important one based on its established role in bacterial phagocytosis and the maintenance of immune homeostasis. Accordingly, we have deciphered how this signaling pathway differentiates between pathogenic and non‐pathogenic bacteria. Wnt5A signaling results in both uptake and killing of pathogenic bacteria including Pseudomonas sp., Streptococcus sp. and E. coli sp. independent of species specificity through cytoskeletal modulations that involve Rac1/Dvl1 activation. However, some laboratory bacterial strains such as E.coli DH5α and E.coli K12MG1655, that lack pathogenicity islands get internalized but not killed by Wnt5A signaling (Figure 1). In comparison to the non‐pathogenic bacterial strains the pathogenic bacterial strains result in decrease of total cellular F‐actin in the host macrophage (Figure 2). Revamping of Wnt5A signaling not only aids in recovery of the host cytoskeletal actin but also helps in containment of the infection. As for the non‐pathogenic strains, Wnt5A signaling aided uptake and sustenance of the bacteria inside the host correlate with increased actin polymerization around bacteria containing phagosomes. Interestingly, disruption of Wnt5A dependent actin polymerization in macrophages by actin polymerization inhibitors leads to increased infection by the pathogenic bacteria but killing of the non‐pathogenic lab strains (Figure 2). Preliminary observations suggest that the relation of some host commensals with Wnt5A signaling is similar to that of the non‐pathogenic bacteria. Our experimental data suggest that the interplay between Wnt5A induced actin polymerization and the pathogenic potential of incumbent bacteria is a major factor determining the outcome of the host‐bacteria interactions and bacterial persistence vs. eradication.Support or Funding InformationThis work was supported by funding from DBT, Govt. of India (BT/PR7106/MED/29/639/2012, BT/PR27125/BRB/10/1635/2017) and Institutional funding. SJ and SS were supported by Research Scholar Fellowship from CSIR, Govt. of India.Wnt5A signaling regulates host response to pathogenic and non‐pathogenic bacterial strains.In line with our previous report of Wnt5A signaling mediated killing of Pseudomonas and Streptococcus (Jati et al. Frontiers in immunology, 2018) we observed Wnt5A mediated killing of pathogenic Escherichia coli K1 strain (MOI 10) in both RAW264.7 and peritoneal macrophages (PMϕ) (a,b). But Wnt5A signaling in RAW264.7 and peritoneal macrophages (PMϕ) resulted in enhanced survival of non‐pathogenic bacterial strain Escherichia coli K12MG1655 (MOI 10) (c,d). Data represented as mean ± SEM; *p ≤ 0.05, **p ≤ 0.005, ***p≤ 0.0005. PBS: vehicle control for Wnt5A, MOI: Multiplicity of Infection, CFU: Colony Forming UnitsFigure 1Alteration of cytoskeletal actin in relation to Wnt5A signaling modulates the outcome of host‐bacteria interaction.Infection with pathogenic Escherichia coli K1 strain results in decrease of total cellular F‐actin compared to the non‐pathogenic Escherichia coli K12MG1655 and Escherichia coli DH5α (a). After 3hrs post infection (T3) of pathogenic Escherichia coli K1 the cellular F‐actin level increased in the Wnt5A treated macrophage set (b) compared to vehicle control (PBS). But in case of non‐pathogenic Escherichia coli DH5α there was no such change (b). In presence of Wnt5A, Arp2 inhibitor CK‐666 (Arp2 inh.) induces clearance of non‐pathogenic Escherichia coli K12MG1655 and Escherichia coli DH5α at 3hrs post infection but inhibits the killing of pathogenic Escherichia coli K1 (c). Data represented as mean ± SEM; *p ≤ 0.05, **p≤ 0.005, ***p≤ 0.0005. MOI: Multiplicity of InfectionFigure 2

Pathogenic Puppetry: Manipulation of the Host Actin Cytoskeleton by Chlamydia trachomatis.

The actin cytoskeleton is crucially important to maintenance of the cellular structure, cell motility, and endocytosis. Accordingly, bacterial pathogens often co-opt the actin-restructuring machinery of host cells to access or create a favorable environment for their own replication. The obligate intracellular organism Chlamydia trachomatis and related species exemplify this dynamic: by inducing actin polymerization at the site of pathogen-host attachment, Chlamydiae induce their own uptake by the typically non-phagocytic epithelium they infect. The interaction of chlamydial adhesins with host surface receptors has been implicated in this effect, as has the activity of the chlamydial effector TarP (translocated actin recruitment protein). Following invasion, C. trachomatis dynamically assembles and maintains an actin-rich cage around the pathogen’s membrane-bound replicative niche, known as the chlamydial inclusion. Through further induction of actin polymerization and modulation of the actin-crosslinking protein myosin II, C. trachomatis promotes egress from the host via extrusion of the inclusion. In this review, we present the experimental findings that can inform our understanding of actin-dependent chlamydial pathogenesis, discuss lingering questions, and identify potential avenues of future study.

Ca2+ Flux: Searching for a Role in Efferocytosis of Apoptotic Cells in Atherosclerosis.

In atherosclerosis, macrophages in the arterial wall ingest plasma lipoprotein-derived lipids and become lipid-filled foam cells with a limited lifespan. Thus, efficient removal of apoptotic foam cells by efferocytic macrophages is vital to preventing the dying foam cells from forming a large necrotic lipid core, which, otherwise, would render the atherosclerotic plaque vulnerable to rupture and would cause clinical complications. Ca2+ plays a role in macrophage migration, survival, and foam cell generation. Importantly, in efferocytic macrophages, Ca2+ induces actin polymerization, thereby promoting the formation of a phagocytic cup necessary for efferocytosis. Moreover, in the efferocytic macrophages, Ca2+ enhances the secretion of anti-inflammatory cytokines. Various Ca2+ antagonists have been seminal for the demonstration of the role of Ca2+ in the multiple steps of efferocytosis by macrophages. Moreover, in vitro and in vivo experiments and clinical investigations have revealed the capability of Ca2+ antagonists in attenuating the development of atherosclerotic plaques by interfering with the deposition of lipids in macrophages and by reducing plaque calcification. However, the regulation of cellular Ca2+ fluxes in the processes of efferocytic clearance of apoptotic foam cells and in the extracellular calcification in atherosclerosis remains unknown. Here, we attempted to unravel the molecular links between Ca2+ and efferocytosis in atherosclerosis and to evaluate cellular Ca2+ fluxes as potential treatment targets in atherosclerotic cardiovascular diseases.

RAC1 Inhibitor EHT1864 and Venetoclax Overcome Midostaurin Resistance in Acute Myeloid Leukemia

Introduction Acute Myeloid Leukemia (AML) is a genetically heterogeneous disease characterized by clonal expansion of immature myeloid progenitor cells in the bone marrow (BM). Mutations of the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 30% of AML cases, with Internal Tandem Duplications (ITD) being the most common type of mutation. Several FLT3 specific inhibitors (TKI) have been developed such as quizartinib, crenolanib and midostaurin (recently approved for clinical use). Nevertheless FLT3-ITD is associated with unfavorable prognosis and patients develop drug resistance with the underlying mechanisms remaining largely unexplained. Recently, changes within the actin cytoskeleton were associated with drug resistance development in various cancers. FLT3-ITD mutations are associated with RAC1 activation. RAC1 belongs to the family of RHO GTPases and enhances the actin polymerization by inducing the expression of N-WASP or WAVE2 and ARP2/3 complex. Therefore, we investigated actin cytoskeleton rearrangements through RAC1 activation as a potential mechanism contributing to Midostaurin resistance in AML. Material and methods First, we developed two Midostaurin resistant AML cell lines (MID-RES, MV4-11 and MOLM-13). Single cell measurements of Cell Stiffnes, cell adhesion forces between tumor and HS5 stroma cells and Actin filaments were performed by Atomic Force Microscopy (FluidFM®) and SIM microscopy, respectively. RAC1 activation was measured by RAC1 activation kit provided by Cytoskeleton. FLT3 surface and intracellular expression was measured by Flow cytometry and western blot, respectively. Cell death was analyzed by Annexin/PI staining in flow cytometry. Results The MID-RES cell lines MV4-11/MOLM-13 showed higher FLT3 surface and intracellular expression compared to their MID sensitive parental cells. In line with our expectations, we observed RAC1 activation, as well as an up-regulation of actin polymerization positive regulators such as N-WASP, WAVE2, PFN1 and ARP2/3 complex and the inhibition of actin polymerization negative regulator P-ser3 CFL1 in MID-RES cells. FLT3 receptor knock down by siRNAs reversed the MID resistance and reduced RAC1 activation and actin polymerization inducers expression. Likewise, bioinformatic analysis from publicly available microarray expression data (E-MTAB-3444), confirmed positive correlation between actin polymerization inducers and FLT3 signaling expression in 178 FLT3-ITD (r=0,67) and 461 FLT3 WT(r= 0,57) de novoAML patients. RAC1 induced Actin polymerization positively correlates with actin filaments growth and cell stiffness, which was observed in our MID-RES cells, higher load of actin filaments and increased cell stiffness. The combination between RAC1 specific inhibitor, EHT1864 and Midostaurin synergistically induces cell death in MID-RES cells by arresting cell cycle in G0/G1 phase and activating apoptosis. Beside, this combination reduced the adhesion forces to stroma cells, decreased the expression of PFN1/N-WASP/ARP2 and consequently reduced drastically the number of actin filaments and cell stiffness in MID-RES cells. EHT1864 and Midostaurin (alone and in combination) were not toxic in PBMCs obtained from healthy donors. Interestingly, this combination increase >45 % cell death in cells obtained from refractory FLT3-mutated AML patient (this patient was relapsed (≥ 50% residual blasts in the bone marrow)under Chemotherapy+Midostaurin combination).The specific knock down of PFN1/N-WASP/ARP2 with siRNAs equally reversed the resistance to Midostaurin. Of note, RAC1 regulates the anti-apoptotic BCL2. Indeed, EHT1864 in combination with Midostaurin reduced anti-apoptotic family BCL2/MCL1 expression and increases the pro-apoptotic proteins BAX/PUMA. As expected, our MID-RES cells showed higher sensitivity to BCL2 inhibitor Venetoclax, than their parental cells. The combinations EHT1864+venetoclax, venetoclax+midostaurin and venetoclax+Midostaurin+EHT1864 synergistically induced cell death in MID-RES cells. Conclusion Actin polymerization inducers N-WASP, ARP2/3 complex and PFN1 may provide a valuable approach to overcome Midostaurin resistance in AML. Our data further suggest that the addition of BCL2 inhibition through EHT1864 and venetoclax could represent an interesting strategy to potentiate the activity of Midostaurin in FLT3 mutated AML. Disclosures Duell: Regeneron Pharmaceuticals, Inc.: Research Funding. Rosenwald:MorphoSys: Consultancy.

Carbon nanotubes as molecular transporters to study a new mechanism for molecular entry into the cell nucleus using actin polymerization force.

The transport of macromolecules into the cell nucleus occurs through nuclear pore complexes (NPCs) and is mediated by cellular receptors. Recently, a novel mechanism of nuclear entry, in which actin polymerization provides a propulsive force driving the transport through the NPC, has been proposed. This mechanism is used by the nucleocapsid from baculovirus, one of the largest viruses to replicate in the nucleus of their host cells, which crosses the NPC and enters the nucleus independently of cellular receptors. The baculovirus nucleocapsid contains a protein that hijacks the cellular actin polymerization machinery to assemble actin filaments that propel the nucleocapsid through the host cell cytoplasm. In this study, we functionalized carbon nanotubes by covalently attaching a protein domain responsible for inducing actin polymerization and investigated their nuclear entry. We found that the functionalized carbon nanotubes were able to enter the cell nucleus under permissive conditions for actin polymerization, but not when this process was inhibited. We conclude that the mechanical force generated by actin polymerization can drive cargo entry into the cell nucleus. Our results support a novel force-driven mechanism for molecular entry into the cell nucleus.

Vav mediates Ras stimulation by direct activation of the GDP/GTP exchange factor Ras GRP1.

Here we describe a new signaling cross-talk between the Vav/Rac1 and Ras pathways that is established through the stimulation of RasGRP1, an exchange factor for Ras subfamily GTPases. This interaction is crucial for Ras activation in lymphoid cells, since this GTPase cannot become activated in the absence of Vav proteins. The activation of RasGRP1 requires both the generation of diacylglycerol via phospho lipase C-γ and the induction of actin polymerization, two responses induced by Vav and Rac1 that facilitate the translocation of RasGRP1 to juxtamembrane areas of the cell. Consistent with this, the cross-talk can be activated by tyrosine-phosphorylated wild-type Vav, oncogenic Vav and constitutively active Rac1. Conversely, Ras activation can be blocked in lymphocytes and ectopic systems using inhibitors affecting either phospholipase C-γ or F-actin polymerization. These results indicate that a relay mechanism exists in lymphoid and other cells helping in the generation of robust signaling responses by the Rac/Rho and Ras pathways upon receptor engagement.

Abstract B183: LFA-1 signals via Crk adapter proteins to induce actin-dependent T-cell migration and mechanosensing

Abstract Allogeneic hematopoietic stem cell transplants (HSCT) are used to treat many malignancies, but the prevalence of graft-versus-host disease (GvHD) limits their overall success. Manipulating T-cell trafficking has emerged as an effective countermeasure, yet downstream integrin signaling pathways have yet to be targeted. We previously found that T-cells lacking the adapter proteins Crk and CrkL exhibit a robust antitumor response while causing little GvHD. We now find that T-cells from mice lacking Crk adapter proteins exhibit defects in LFA-1/ICAM-1 induced actin polymerization, leading edge formation, 2D migration, and integrin-mediated mechanosensing. Under shear flow, Crk/CrkL deficient T-cells fail to migrate upstream on ICAM-1 but migrate normally on VCAM-1, suggesting these integrin ligands relay different outside-in signals. Analysis of LFA-1 signaling reveals that Crk protein expression is required for phosphorylation of c-Cbl and its subsequent interaction with the PI3K subunit p85. Through this mechanism, Crk proteins promote PI3K activity and cytoskeletal remodeling downstream of LFA-1 engagement. Interestingly, this signaling pathway was largely specific to the LFA-1/ICAM-1 interaction, as WT-cells plated on VCAM-1 (which binds and signals through VLA-4) failed to induce high levels of phospho-Cbl, showed diminished PI3K activity, and did not migrate with a defined actin-rich leading edge. Finally, we show that knocking out CrkL alone is sufficient to alleviate GvHD, pointing toward unique roles of the Crk isoforms in T-cell biology. Together, these studies identify key signaling differences downstream of β2 vs β1 integrins that drive T-cell migratory behavior, and identify CrkL as an important factor during GvHD. Importantly, these data provide insight into integrin signaling that could be used to manipulate T-cell trafficking. Citation Format: Nathan H. Roy, Joanna L. MacKay, Alexander Buffone Jr., Krista Newell, Tanner F. Robertson, Sangya Agarwal, Mobin Karimi, Daniel A Hammer, Janis K. Burkhardt. LFA-1 signals via Crk adapter proteins to induce actin-dependent T-cell migration and mechanosensing [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B183.

Formyl peptide receptor-1 activation exerts a critical role for the dynamic plasticity of arteries via actin polymerization

Several human diseases, include cancer and stroke are characterized by changes in immune system activation and vascular contractility. However, the mechanistic foundation of a vascular immuno-physiology network is still largely unknown. Formyl peptide receptor-1 (FPR-1), which plays a vital role in the function of the innate immune system, is widely expressed in arteries, but its role in vascular plasticity is unclear. We questioned why a receptor that is crucial for immune defense, and cell motility in leukocytes, would be expressed in vascular smooth muscle cells (VSMCs). We hypothesized that activation of FPR-1 in arteries is important for the temporal reorganization of actin filaments, and consequently, changes in vascular function, similar to what is observed in neutrophils. To address our hypothesis, we used FPR-1 knockout and VSMCs lacking FPR-1. We observed that FPR-1 activation induces actin polymerization in wild type VSMCs. Absence of FPR-1 in the vasculature significantly decreased vascular contraction and induced loss of myogenic tone to elevated intraluminal pressures via disruption of actin polymerization. Actin polymerization activator ameliorated these responses. In conclusion, we have established a novel role for FPR-1 in VSMC contractility and motility, similar to the one observed in sentinel cells of the innate immune system. This discovery is fundamental for vascular immuno-pathophysiology, given that FPR-1 in VSMCs not only functions as an immune system receptor, but it also has an important role for the dynamic plasticity of arteries.

Nano-Antagonist Alleviates Inflammation and Allows for MRI of Atherosclerosis.

Specific targeting of inflammation remains a challenge in many pathologies, because of the necessary balance between host tolerance and efficacious inflammation resolution. Here, we discovered a short, 4-mer peptide which possesses antagonist properties towards CC chemokine receptor 2 (CCR2), but only when displayed on the surface of lipid nanoparticles. According to BLAST analysis, this peptide motif is a common repeating fragment in a number of proteins of the CC chemokine family, which are key players in the inflammatory response. In this study, self-assembled, peptide-conjugated nanoparticles (CCTV) exhibited typical properties of CCR2 antagonism, including affinity to the CCR2 receptor, inhibition of chemotactic migration of primary monocytes, and prevention from CC chemokine ligand 2 (CCL2)-induced actin polymerization. Furthermore, CCTV ameliorated NFkB activation and downregulated the secondary, but not the primary, inflammatory response in cultured macrophages. When conjugated with gadolinium or europium cryptates, CCTV enabled targeted imaging (via magnetic resonance imaging and time-resolved fluorescence) of atherosclerosis, a chronic inflammatory condition in which the CCL2/CCR2 axis is highly dysfunctional. CCTV targeted CCR2hiLy6Chi inflammatory monocytes in blood and the atherosclerotic plaque, resulting in cell-specific transcriptional downregulation of key inflammatory genes. Finally, CCTV generated pronounced inflammasome inactivation, likely mediated through reactive oxygen species scavenging and downregulation of NLRP3. In summary, our work demonstrates for the first time that a short peptide fragment presented on a nanoparticle surface exhibit potent receptor-targeted antagonist effects, which are not seen with the peptide alone. Unlike commonly used cargo-carrying, vector-directed drug delivery vehicles, CCTV nanoparticles may act as therapeutics/theranostics themselves, particularly in inflammatory conditions with CCL2/CCR2 pathogenesis, including cardiovascular disease and cancer.

Regulation of gastric smooth muscle contraction via Ca2+-dependent and Ca2+-independent actin polymerization.

In gastrointestinal smooth muscle, acetylcholine induced muscle contraction is biphasic, initial peak followed by sustained contraction. Contraction is regulated by phosphorylation of 20 kDa myosin light chain (MLC) at Ser19, interaction of actin and myosin, and actin polymerization. The present study characterized the signaling mechanisms involved in actin polymerization during initial and sustained muscle contraction in response to muscarinic M3 receptor activation in gastric smooth muscle cells by targeting the effectors of initial (phospholipase C (PLC)-β/Ca2+ pathway) and sustained (RhoA/focal adhesion kinase (FAK)/Rho kinase pathway) contraction. The initial Ca2+ dependent contraction and actin polymerization is mediated by sequential activation of PLC-β1 via Gαq, IP3 formation, Ca2+ release and Ca2+ dependent phosphorylation of proline-rich-tyrosine kinase 2 (Pyk2) at Tyr402. The sustained Ca2+ independent contraction and actin polymerization is mediated by activation of RhoA, and phosphorylation of FAK at Tyr397. Both phosphorylation of Pyk2 and FAK leads to phosphorylation of paxillin at Tyr118 and association of phosphorylated paxillin with the GEF proteins p21-activated kinase (PAK) interacting exchange factor α, β (α and β PIX) and DOCK 180. These GEF proteins stimulate Cdc42 leading to the activation of nucleation promoting factor N-WASP (neuronal Wiskott-Aldrich syndrome protein), which interacts with actin related protein complex 2/3 (Arp2/3) to induce actin polymerization and muscle contraction. Acetylcholine induced muscle contraction is inhibited by actin polymerization inhibitors. Thus, our results suggest that a novel mechanism for the regulation of smooth muscle contraction is mediated by actin polymerization in gastrointestinal smooth muscle which is independent of MLC20 phosphorylation.

G-protein-coupled formyl peptide receptors play a dual role in neutrophil chemotaxis and bacterial phagocytosis.

A dogma of innate immunity is that neutrophils use G-protein–coupled receptors (GPCRs) for chemoattractant to chase bacteria through chemotaxis and then use phagocytic receptors coupled with tyrosine kinases to destroy opsonized bacteria via phagocytosis. Our current work showed that G-protein–coupled formyl peptide receptors (FPRs) directly mediate neutrophil phagocytosis. Mouse neutrophils lacking formyl peptide receptors (Fpr1/2–/–) are defective in the phagocytosis of Escherichia coli and the chemoattractant N-formyl-Met-Leu-Phe (fMLP)-coated beads. fMLP immobilized onto the surface of a bead interacts with FPRs, which trigger a Ca2+ response and induce actin polymerization to form a phagocytic cup for engulfment of the bead. This chemoattractant GPCR/Gi signaling works independently of phagocytic receptor/tyrosine kinase signaling to promote phagocytosis. Thus, in addition to phagocytic receptor-mediated phagocytosis, neutrophils also utilize the chemoattractant GPCR/Gi signaling to mediate phagocytosis to fight against invading bacteria.

An endogenous chemorepellent directs cell movement by inhibiting pseudopods at one side of cells.

Eukaryotic chemoattraction signal transduction pathways, such as those used by Dictyostelium discoideum to move toward cAMP, use a G protein–coupled receptor to activate multiple conserved pathways such as PI3 kinase/Akt/PKB to induce actin polymerization and pseudopod formation at the front of a cell, and PTEN to localize myosin II to the rear of a cell. Relatively little is known about chemorepulsion. We previously found that AprA is a chemorepellent protein secreted by Dictyostelium cells. Here we used 29 cell lines with disruptions of cAMP and/or AprA signal transduction pathway components, and delineated the AprA chemorepulsion pathway. We find that AprA uses a subset of chemoattraction signal transduction pathways including Ras, protein kinase A, target of rapamycin (TOR), phospholipase A, and ERK1, but does not require the PI3 kinase/Akt/PKB and guanylyl cyclase pathways to induce chemorepulsion. Possibly as a result of not using the PI3 kinase/Akt/PKB pathway and guanylyl cyclases, AprA does not induce actin polymerization or increase the pseudopod formation rate, but rather appears to inhibit pseudopod formation at the side of cells closest to the source of AprA.

Fasudil inhibits actin polymerization and collagen synthesis and induces apoptosis in human urethral scar fibroblasts via the Rho/ROCK pathway.

PurposeTo examine the effects and mechanism of action of fasudil on cytoskeletal polymerization, collagen synthesis, and apoptosis in fibroblasts derived from human urethral scar tissue.Materials and methodsFibroblasts treated with or without transforming growth factor β1 (TGF-β1, 10 ng/mL) were incubated with fasudil (12.5, 25, 50 μmol/L) for 24 hours. Quantitative real-time polymerase chain reaction and Western blotting were used to determine the expression of Arp2, Arp3, WASP, and WAVE2. Collagen I and III protein levels were also evaluated by Western blotting. The filamentous actin cytoskeleton was examined by immunofluorescence and epifluorescence microscopy. An Annexin V-FITC/PI staining assay was used to investigate apoptosis.ResultsTGF-β1-dependent induction of actin polymerization and collagen synthesis and promotion of apoptosis were dose dependent. When compared with untreated controls, fasudil significantly decreased the expression of Arp2, Arp3, WASP, WAVE2, Collagen I, and Collagen III in cells treated with or without TGF-β1. Fasudil also promoted apoptosis in cells, irrespective of TGF-β1 treatment.ConclusionIrrespective of TGF-β1 activation status, fasudil suppressed actin polymerization and collagen synthesis and induced apoptosis in human urethral scar fibroblasts via the Rho/ROCK signaling pathway.

Abstract 114: Increased Circulating Levels of Mitochondrial N-Formyl Peptides Leads to Vascular Dysfunction and High Blood Pressure in Spontaneously Hypertensive Rats

Aims: The most powerful signaling pathway that induces actin polymerization and movement in neutrophils is due to formyl peptide receptor (FPR) activation. FPR was originally identified by its ability to bind bacterial N-formyl peptides. Mitochondria carry hallmarks of their bacterial ancestry and one of these hallmarks is that this organelle uses an N-formyl-methionyl-tRNA as an initiator of protein synthesis. Consequently, mitochondrial N-formyl peptides (F-MITs) are recognized by FPR. Recently, we have observed that FPR is expressed in endothelial and vascular smooth muscle cells (VSMC). We hypothesized that increased plasma levels of F-MITs activates FPR and contributes to microvascular damage and hypertension. Methods: Male SHR (12-week old) were treated for 10 days with FPR antagonist cocktail: Cyclosporin H (CsH, 0.1 mg/kg/day i.p. ) and WRW4 (0.1 mg/kg/day i.p. ) or vehicle. Blood pressure (BP) was measured by telemetry. We also treated Wistar rats (12 week old) with F-MITs (formylated peptide corresponding to the NH2-terminus of mitochondria ND6; 0.02 mg/rat/day i.p. ) or non-formylated peptides (control) for 10 days. Blood was collected to measure F-MITs. Intrarenal arteries and mesenteric resistance arteries (MRA) were isolated and mounted in a wire myograph. Wistar VSMCs were treated with F-MITs (20 min, 10 μM) or control in the presence or absence of FPR antagonists to evaluate actin polymerization. T-test *p<0.05; n=5-6. Results and Conclusion: F-MITs plasma levels were increased in SHR (1.7-fold* vs. Wistar). FPR blockade decreased BP (SHR + vehicle: 151 ± 7.2 vs. SHR+CsH+WRW4: 138 ± 0.4* mmHg) and ameliorated acetylcholine-induced relaxation (Intrarenal; Emax: SHR+vehicle: 10 ± 5 vs. SHR+CsH+WRW4: 28 ± 6* %); (MRA; Emax: SHR+vehicle: 74±10 vs. SHR+CsH+WRW4: 99±0.6* %). F-MITs infusion increased BP in Wistar rats (Control: 133 ± 1.9 vs. F-MITs: 145 ± 4.7* mmHg) and decreased acetylcholine-induced relaxation (EC50; MRA: Control: -8.765 ± 0.05 vs. F-MIT: -8.348 ± 0.08*) (Emax; Intrarenal: Control: 63±4 vs. F-MIT: 46 ± 3* %). F-MITs induced VSMCs actin polymerization (1.8 fold* vs. control) and FPR blockers abolished this result. In conclusion, FMITs play a role in vascular dysfunction and hypertension via FPR activation.

Farnesoid X Receptor Activation Enhances Transforming Growth Factor β-Induced Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma Cells.

Farnesoid X receptor (FXR) is a receptor for bile acids and plays an important role in the regulation of bile acid metabolism in the liver. Although FXR has been shown to affect hepatocarcinogenesis through both direct and indirect mechanisms, potential roles of FXR in epithelial–mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) remain unclear. We examined the effect of several FXR ligands on EMT-related morphological changes in HCC cell lines, such as HuH-7 and Hep3B cells. FXR agonists (chenodeoxycholic acid, GW4064, and obeticholic acid)—but not an antagonist (guggulsterone)—induced actin polymerization and expression of N-cadherin and phosphorylated focal adhesion kinase, although they were less effective than transforming growth factor β (TGF-β). FXR agonist treatment enhanced TGF-β-induced EMT morphologic changes and FXR antagonist inhibited the effect of TGF-β. Thus, FXR activation enhances EMT in HCC and FXR antagonists may be EMT-suppressing drug candidates.