Dynamic in situ confinement triggers ligand-free neuropeptide receptor signaling

Membrane receptors are central to cell-cell communication. Receptor clustering at the plasma membrane modulates physiological responses, and microscale receptor organization is critical for downstream signaling. Spatially restricted cluster formation of the neuropeptide Y2 hormone receptor (Y2R) was observed in vivo; however, the relevance of this confinement is not fully understood. Here, we controlled Y2R clustering in situ by a multivalent chelator nanotool in prestructured matrices. Fast Y2R enrichment in microscale arrays triggered a ligand-independent downstream signaling determined by an increase in cytosolic calcium, cell spreading and migration. We reveal that ligand-independent signaling by confinement differs from ligand-induced activation in the recruitment of arrestin-3 as downstream effector, which was recruited to confined regions only in presence of the ligand. The employed multivalent nanotool facilitated a dynamic receptor enrichment with high exchange in the confined regions, comparable to microscale condensates. This concept enables in situ organization of membrane receptors and the exploration of ligand-independent receptor signaling.

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Exosomal MicroRNAs Derived from Human Amniotic Epithelial Cells Accelerate Wound Healing by Promoting the Proliferation and Migration of Fibroblasts

Stem Cells International ◽

10.1155/2018/5420463 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10 ◽

Cited By ~ 11

Author(s):

Bin Zhao ◽

Xiaodong Li ◽

Xiaomin Shi ◽

Xueqin Shi ◽

Wei Zhang ◽

...

Keyword(s):

Wound Healing ◽

Epithelial Cells ◽

Cell Communication ◽

Rnase A ◽

Proteinase K ◽

Amniotic Epithelial Cells ◽

Human Amniotic Epithelial Cells ◽

And Migration ◽

Proliferation And Migration

Previous work in our laboratory demonstrated that exosomes derived from human amniotic epithelial cells (hAECs) accelerated wound healing by promoting the proliferation and migration of fibroblasts. It is reported that exosomes, which are carriers of the microRNAs (miRNAs) and proteins, play an important role in the regulation of cell-to-cell communication. However, it is still unclear precisely which molecule or which group of molecules carried within hAEC-derived exosomes (hAEC-Exos) mediated wound healing. Here, we explored purified hAEC-Exos together with either proteinase K (PROse) or RNase A on the effect of fibroblasts and cutaneous wound healing. Our experiments demonstrated that hAEC-Exos were positive for exosomal markers CD9, CD63, and CD81. Also, we found that hAEC-Exos could be internalized by fibroblasts and then stimulated cell migration and proliferation. However, the promotive effect of hAEC-Exos was abolished by pretreating hAEC-Exos with RNase A, not PROse. Importantly, in vivo wound healing assay showed that local injection of hAEC-Exos or PROse pretreated hAEC-Exos at skin wounds significantly accelerated wound healing. Our findings revealed an important role of exosomal miRNAs in wound healing.

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Expression of integrin receptors and their role in adhesion, spreading and migration of normal human melanocytes

Journal of Cell Science ◽

10.1242/jcs.105.1.179 ◽

1993 ◽

Vol 105 (1) ◽

pp. 179-190 ◽

Cited By ~ 2

Author(s):

G. Zambruno ◽

P.C. Marchisio ◽

A. Melchiori ◽

S. Bondanza ◽

R. Cancedda ◽

...

Keyword(s):

Wound Healing ◽

Membrane Surface ◽

Integrin Receptors ◽

Human Melanocytes ◽

Normal Human ◽

And Migration ◽

Alpha V Beta 3

Integrin receptors of human melanocytes in vivo and of melanocytes isolated and cultured from in vitro reconstituted normal human epidermis were investigated. Melanocytes were studied by high-resolution immunocytochemistry of in situ epidermis and were found to expose only the integrin subunits alpha 3, alpha 6, alpha v and beta 1 on their plasma membrane surface. Instead, cultured normal melanocytes expressed alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1 and alpha v beta 3, which were immunoprecipitated from both metabolically and surface-labeled cells. Beta 1 integrins were diffused on the adhesion surface, while alpha v beta 3 was clustered in focal contacts both in control cells and upon dendrite induction with phorbol 12-myristate 13-acetate (PMA). The functional roles of integrins were studied in vitro by cell adhesion, spreading and migration assays. The sum of the data indicated that, in normal human melanocytes: (i) adhesion to defined substrata is mainly mediated by specific beta 1 integrins; (ii) spreading is mainly modulated by alpha v beta 3; (iii) the beta 1 and beta 3 heterodimers cooperate in regulating migration. The in vitro expression of two integrins (alpha v beta 3 and alpha 5 beta 1) that are not exposed in situ, and their role in the spreading and migratory properties of melanocytes, strongly suggest that they are involved in regenerating a normally pigmented epidermis during wound healing by controlling melanocyte spreading and migration over a provisional matrix. Tumor promoters, such as PMA, selectively increased the expression of alpha 3 beta 1. We suggest that this integrin might be involved in melanocyte migration on the newly formed basement membrane during wound healing as well as in intercellular recognition of adjacent keratinocytes.

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Calmodulin kinase II is required for angiotensin II-mediated vascular smooth muscle hypertrophy

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01014.2009 ◽

2010 ◽

Vol 298 (2) ◽

pp. H688-H698 ◽

Cited By ~ 52

Author(s):

Hui Li ◽

Weiwei Li ◽

Arun K. Gupta ◽

Peter J. Mohler ◽

Mark E. Anderson ◽

...

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Muscle Hypertrophy ◽

Central Feature ◽

Ang Ii ◽

Downstream Signaling ◽

And Migration ◽

Medial Hypertrophy

Despite our understanding that medial smooth muscle hypertrophy is a central feature of vascular remodeling, the molecular pathways underlying this pathology are still not well understood. Work over the past decade has illustrated a potential role for the multifunctional calmodulin-dependent kinase CaMKII in smooth muscle cell contraction, growth, and migration. Here we demonstrate that CaMKII is enriched in vascular smooth muscle (VSM) and that CaMKII inhibition blocks ANG II-dependent VSM cell hypertrophy in vitro and in vivo. Specifically, systemic CaMKII inhibition with KN-93 prevented ANG II-mediated hypertension and medial hypertrophy in vivo. Adenoviral transduction with the CaMKII peptide inhibitor CaMKIIN abrogated ANG II-induced VSM hypertrophy in vitro, which was augmented by overexpression of CaMKII-δ2. Finally, we identify the downstream signaling components critical for ANG II- and CaMKII-mediated VSM hypertrophy. Specifically, we demonstrate that CaMKII induces VSM hypertrophy by regulating histone deacetylase 4 (HDAC4) activity, thereby stimulating activity of the hypertrophic transcription factor MEF2. MEF2 transcription is activated by ANG II in vivo and abrogated by the CaMKII inhibitor KN-93. Together, our studies identify a complete pathway for ANG II-triggered arterial VSM hypertrophy and identify new potential therapeutic targets for chronic human hypertension.

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Silencing of Transforming Growth Factor-β1 In situ by RNA Interference for Breast Cancer: Implications for Proliferation and Migration In vitro and Metastasis In vivo

Clinical Cancer Research ◽

10.1158/1078-0432.ccr-07-4604 ◽

2008 ◽

Vol 14 (15) ◽

pp. 4961-4970 ◽

Cited By ~ 41

Author(s):

Lakisha D. Moore ◽

Tatyana Isayeva ◽

Gene P. Siegal ◽

Selvarangan Ponnazhagan

Keyword(s):

Breast Cancer ◽

Rna Interference ◽

Growth Factor ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β1 ◽

And Migration ◽

Proliferation And Migration

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Phage-Displayed Peptides for Targeting Tyrosine Kinase Membrane Receptors in Cancer Therapy

Viruses ◽

10.3390/v13040649 ◽

2021 ◽

Vol 13 (4) ◽

pp. 649

Author(s):

Annamaria Aloisio ◽

Nancy Nisticò ◽

Selena Mimmi ◽

Domenico Maisano ◽

Eleonora Vecchio ◽

...

Keyword(s):

Cancer Therapy ◽

Tyrosine Kinase ◽

Phage Display ◽

Tyrosine Kinases ◽

Peptide Ligands ◽

Membrane Receptors ◽

Downstream Signaling ◽

Cancer Pathogenesis

Receptor tyrosine kinases (RTKs) regulate critical physiological processes, such as cell growth, survival, motility, and metabolism. Abnormal activation of RTKs and relative downstream signaling is implicated in cancer pathogenesis. Phage display allows the rapid selection of peptide ligands of membrane receptors. These peptides can target in vitro and in vivo tumor cells and represent a novel therapeutic approach for cancer therapy. Further, they are more convenient compared to antibodies, being less expensive and non-immunogenic. In this review, we describe the state-of-the-art of phage display for development of peptide ligands of tyrosine kinase membrane receptors and discuss their potential applications for tumor-targeted therapy.

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Targeting PKC in multiple myeloma: in vitro and in vivo effects of the novel, orally available small-molecule inhibitor enzastaurin (LY317615.HCl)

Blood ◽

10.1182/blood-2006-08-042747 ◽

2006 ◽

Vol 109 (4) ◽

pp. 1669-1677 ◽

Cited By ~ 81

Author(s):

Klaus Podar ◽

Marc S. Raab ◽

Jing Zhang ◽

Douglas McMillin ◽

Iris Breitkreutz ◽

...

Keyword(s):

Multiple Myeloma ◽

Xenograft Model ◽

Multidrug Resistant ◽

The Novel ◽

Pkc Inhibitor ◽

Downstream Signaling ◽

Synergistic Cytotoxicity ◽

And Migration

Abstract In multiple myeloma (MM) protein kinase C (PKC) signaling pathways have been implicated in cell proliferation, survival, and migration. Here we investigated the novel, orally available PKC-inhibitor enzastaurin for its anti-MM activity. Enzastaurin specifically inhibits phorbol ester–induced activation of PKC isoforms, as well as phosphorylation of downstream signaling molecules MARCKS and PKCμ. Importantly, it also inhibits PKC activation triggered by growth factors and cytokines secreted by bone marrow stromal cells (BMSCs), costimulation with fibronectin, vascular endothelial growth factor (VEGF), or interleukin-6 (IL-6), as well as MM patient serum. Consequently, enzastaurin inhibits proliferation, survival, and migration of MM cell lines and MM cells isolated from multidrug-resistant patients and overcomes MM-cell growth triggered by binding to BMSCs and endothelial cells. Importantly, strong synergistic cytotoxicity is observed when enzastaurin is combined with bortezomib and moderate synergistic or additive effects when combined with melphalan or lenalidomide. Finally, tumor growth, survival, and angiogenesis are abrogated by enzastaurin in an in vivo xenograft model of human MM. Our results therefore demonstrate in vitro and in vivo efficacy of the orally available PKC inhibitor enzastaurin in MM and strongly support its clinical evaluation, alone or in combination therapies, to improve outcome in patients with MM.

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Reexpression of Let-7g MicroRNA Inhibits the Proliferation and Migration via K-Ras/HMGA2/Snail Axis in Hepatocellular Carcinoma

BioMed Research International ◽

10.1155/2014/742417 ◽

2014 ◽

Vol 2014 ◽

pp. 1-12 ◽

Cited By ~ 10

Author(s):

Ke-ji Chen ◽

Ying Hou ◽

Kui Wang ◽

Jun Li ◽

Yong Xia ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Epithelial Mesenchymal Transition ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

Mtt Method ◽

Carcinoma Cell Lines ◽

And Migration

Let-7 family microRNAs have been reported to be downregulated in human hepatocellular carcinoma in comparison with normal hepatic tissues. Among them, let-7g was identified as the lowest expression using real-time RT-PCR. However, the mechanism by which let-7g works in hepatocellular carcinoma remains unknown. Here, in our present study, we have had let-7g reexpressedin vitroin hepatocellular carcinoma cell lines MHCC97-H and HCCLM3 via transfection. The proliferation after reexpression of let-7g was assayed using MTT method; the migration and invasion after restoration were detected by wound-healing and Transwell assay, respectively. We found using Western-blotting that let-7g can regulate epithelial-mesenchymal transition (EMT) by downregulating K-Ras and HMGA2A after reexpresssion. Xenografted nude mice were used to observe whether or not reexpression of let-7g could have potential therapeutic ability.In vivo, to observe the association with let-7g expression and overall prognosis, 40 paired cases of hepatocellular carcinoma were analyzed using in situ hybridization (ISH). It was found that reexpression of let-7g can inhibit the proliferation, migration, and invasion significantly, and that low expression of let-7g was significantly associated with poorer overall survival. Taken together, let-7g could be used as a promising therapeutic agentin vivoin the treatment of hepatocellular carcinoma at the earlier stage.

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Manipulating leukocyte interactions in vivo through optogenetic chemokine release

Blood ◽

10.1182/blood-2015-11-684852 ◽

2016 ◽

Vol 127 (23) ◽

pp. e35-e41 ◽

Cited By ~ 14

Author(s):

Milka Sarris ◽

Romain Olekhnovitch ◽

Philippe Bousso

Keyword(s):

Cell Migration ◽

Immune Cell ◽

Cell Communication ◽

In Situ Studies ◽

Key Points ◽

Immune Cell Migration

Key Points We report a method to optogenetically control the release of soluble mediators, such as chemokines, and influence immune cell migration. This approach is applicable to a variety of secreted ligands and can facilitate dynamic, in situ studies of immune cell communication.

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αvβ3 integrin spatially regulates VASP and RIAM to control adhesion dynamics and migration

The Journal of Cell Biology ◽

10.1083/jcb.200912014 ◽

2010 ◽

Vol 189 (2) ◽

pp. 369-383 ◽

Cited By ~ 63

Author(s):

Daniel C. Worth ◽

Kairbaan Hodivala-Dilke ◽

Stephen D. Robinson ◽

Samantha J. King ◽

Penny E. Morton ◽

...

Keyword(s):

Regulatory Protein ◽

Focal Adhesions ◽

Αvβ3 Integrin ◽

Β1 Integrin ◽

Downstream Signaling ◽

Β3 Integrin ◽

And Migration ◽

2D And 3D

Integrins are fundamental to the control of protrusion and motility in adherent cells. However, the mechanisms by which specific members of this receptor family cooperate in signaling to cytoskeletal and adhesion dynamics are poorly understood. Here, we show that the loss of β3 integrin in fibroblasts results in enhanced focal adhesion turnover and migration speed but impaired directional motility on both 2D and 3D matrices. These motility defects are coupled with an increased rate of actin-based protrusion. Analysis of downstream signaling events reveals that loss of β3 integrin results in a loss of protein kinase A–dependent phosphorylation of the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP). Dephosphorylated VASP in β3-null cells is preferentially associated with Rap1-GTP–interacting adaptor molecule (RIAM) both in vitro and in vivo, which leads to enhanced formation of a VASP–RIAM complex at focal adhesions and subsequent increased binding of talin to β1 integrin. These data demonstrate a novel mechanism by which αvβ3 integrin acts to locally suppress β1 integrin activation and regulate protrusion, adhesion dynamics, and persistent migration.

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