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Scientific Reports Jan 2022Following its association with dyslexia in multiple genetic studies, the KIAA0319 gene has been extensively investigated in different animal models but its function in...
Following its association with dyslexia in multiple genetic studies, the KIAA0319 gene has been extensively investigated in different animal models but its function in neurodevelopment remains poorly understood. We developed the first human cellular knockout model for KIAA0319 in RPE1 retinal pigment epithelia cells via CRISPR-Cas9n to investigate its role in processes suggested but not confirmed in previous studies, including cilia formation and cell migration. We observed in the KIAA0319 knockout increased cilia length and accelerated cell migration. Using Elastic Resonator Interference Stress Microscopy (ERISM), we detected an increase in cellular force for the knockout cells that was restored by a rescue experiment. Combining ERISM and immunostaining we show that RPE1 cells exert highly dynamic, piconewton vertical pushing forces through actin-rich protrusions that are surrounded by vinculin-rich pulling sites. This protein arrangement and force pattern has previously been associated to podosomes in other cells. KIAA0319 depletion reduces the fraction of cells forming these actin-rich protrusions. Our results suggest an involvement of KIAA0319 in cilia biology and cell-substrate force regulation.
Topics: Actins; CRISPR-Cas Systems; Cell Communication; Cell Line; Cell Movement; Cilia; Humans; Microscopy, Interference; Models, Genetic; Nerve Tissue Proteins; Podosomes; Retinal Pigment Epithelium; Vinculin
PubMed: 35031635
DOI: 10.1038/s41598-021-04539-3 -
International Journal of Molecular... Dec 2021Erosive destruction of joint structures is a critical event in the progression of rheumatoid arthritis (RA), in which fibroblast-like synoviocytes (FLS) are the primary...
Erosive destruction of joint structures is a critical event in the progression of rheumatoid arthritis (RA), in which fibroblast-like synoviocytes (FLS) are the primary effectors. We previously reported that the ability of RA FLS to degrade extracellular matrix (ECM) components depends on the formation of actin-rich membrane protrusions, called invadosomes, through processes that remain elusive. 14-3-3η belongs to a family of scaffolding proteins involved in a wide range of cellular functions, and its expression is closely related to joint damage and disease activity in RA patients. In this study, we sought to assess the role of 14-3-3η in joint damage by examining its contribution to the invadosome formation phenotype of FLS. Using human primary FLS, we show that 14-3-3η expression is closely associated with their ability to form invadosomes. Furthermore, knockdown of 14-3-3η using shRNAs decreases the level of invadosome formation in RA FLS, whereas addition of the recombinant protein to FLS from healthy individuals promotes their formation. Mechanistic studies suggest that 14-3-3η regulates invadosome formation by increasing Snail expression, a mechanism that involves nuclear exclusion of the transcription repressor FOXO3. Our results implicate the 14-3-3η-FOXO3-Snail axis in promoting the aggressive ECM-degrading phenotype of RA FLS, and suggest a role for this scaffolding protein in cartilage degradation.
Topics: 14-3-3 Proteins; Arthritis, Rheumatoid; Cells, Cultured; Fibroblasts; Forkhead Box Protein O3; Humans; Podosomes; Recombinant Proteins; Snail Family Transcription Factors; Synovial Membrane; Synoviocytes
PubMed: 35008549
DOI: 10.3390/ijms23010123 -
FEBS Letters Feb 2022PI3Kβ is required for invadopodia-mediated matrix degradation by breast cancer cells. Invadopodia maturation requires GPCR activation of PI3Kβ and its coupling to...
PI3Kβ is required for invadopodia-mediated matrix degradation by breast cancer cells. Invadopodia maturation requires GPCR activation of PI3Kβ and its coupling to SHIP2 to produce PI(3,4)P . We now test whether selectivity for PI3Kβ is preserved under conditions of mutational increases in PI3K activity. In breast cancer cells where PI3Kβ is inhibited, short-chain diC8-PIP rescues gelatin degradation in a SHIP2-dependent manner; rescue by diC8-PI(3,4)P is SHIP2-independent. Surprisingly, the expression of either activated PI3Kβ or PI3Kα mutants rescued the effects of PI3Kβ inhibition. In both cases, gelatin degradation was SHIP2-dependent. These data confirm the requirement for PIP conversion to PI(3,4)P for invadopodia function and suggest that selectivity for distinct PI3K isotypes may be obviated by mutational activation of the PI3K pathway.
Topics: Cell Line, Tumor; Cell Movement; Class I Phosphatidylinositol 3-Kinases; Diglycerides; Extracellular Matrix; Female; Gene Expression Regulation; HEK293 Cells; Humans; Mammary Glands, Human; Mutation; Phosphatidylinositol Phosphates; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; Podosomes; Signal Transduction
PubMed: 34990021
DOI: 10.1002/1873-3468.14273 -
There and back again: Intracellular trafficking, release and recycling of matrix metalloproteinases.Biochimica Et Biophysica Acta.... Apr 2022Matrix metalloproteinases are a family of zinc-dependent endopeptidases that are involved in a large variety of proteolytic processes in physiological and pathological... (Review)
Review
Matrix metalloproteinases are a family of zinc-dependent endopeptidases that are involved in a large variety of proteolytic processes in physiological and pathological scenarios, including immune cell surveillance, tissue homeostasis, or tumor cell metastasis. This is based on their ability to cleave a plethora of substrates that include components of the extracellular matrix, but also cell surface-associated and intracellular proteins. Accordingly, a tight regulatory web has evolved that closely regulates spatiotemporal activity of specific MMPs. An often underappreciated mechanism of MMP regulation involves their trafficking to and from specific subcellular sites that require MMP activity only for a certain period. In this review, we focus on the current knowledge of MMP intracellular trafficking, their secretion or surface exposure, as well as their recycling back from the cell surface. We discuss molecular mechanisms that enable these steps, in particular microtubule-dependent motility of vesicles that is driven by molecular motors and directed by vesicle regulatory proteins. Finally, we also point out open questions in the field of MMP motility that may become important in the future.
Topics: Endocytosis; Endoplasmic Reticulum; Extracellular Vesicles; Golgi Apparatus; Humans; Matrix Metalloproteinases; Microtubules; Podosomes; Protein Transport
PubMed: 34973301
DOI: 10.1016/j.bbamcr.2021.119189 -
European Journal of Cell Biology Jan 2022Metastasis remains the main challenge to overcome for treating ovarian cancers. In this study, we investigate the potential role of the Cdc42 GAP StarD13 in the...
Metastasis remains the main challenge to overcome for treating ovarian cancers. In this study, we investigate the potential role of the Cdc42 GAP StarD13 in the modulation of cell motility, invasion in ovarian cancer cells. StarD13 depletion does not affect the 2D motility of ovarian cancer cells. More importantly, StarD13 inhibits matrix degradation, invadopodia formation and cell invasion through the inhibition of Cdc42. StarD13 does not localize to mature TKS4-labeled invadopodia that possess matrix degradation ability, while a Cdc42 FRET biosensor, detects Cdc42 activation in these invadopodia. In fact, StarD13 localization and Cdc42 activation appear mutually exclusive in invadopodial structures. Finally, for the first time we uncover a potential role of Cdc42 in the direct recruitment of TKS4 to invadopodia. This study emphasizes the specific role of StarD13 as a narrow spatial regulator of Cdc42, inhibiting invasion, suggesting the suitability of StarD13 for targeted therapy.
Topics: Adenocarcinoma; Cell Line, Tumor; GTPase-Activating Proteins; Humans; Neoplasm Invasiveness; Podosomes; Tumor Suppressor Proteins; cdc42 GTP-Binding Protein
PubMed: 34958986
DOI: 10.1016/j.ejcb.2021.151197 -
Stem Cell Research & Therapy Dec 2021Enhancer of zeste homolog 2 (EZH2) is a novel oncogene that can specifically trimethylate the histone H3 lysine 27 (H3K27me3) to transcriptionally inhibit the expression...
BACKGROUND
Enhancer of zeste homolog 2 (EZH2) is a novel oncogene that can specifically trimethylate the histone H3 lysine 27 (H3K27me3) to transcriptionally inhibit the expression of downstream tumor-suppressing genes. As a small molecular inhibitor of EZH2, 3-Deazaneplanocin (DZNep) has been widely studied due to the role of tumor suppression. With the roles of epigenetic regulation of bone cells emerged in past decades, the property and molecular mechanism of DZNep on enhancing osteogenesis had been reported and attracted a great deal of attention recently. This study aims to elucidate the role of DZNep on EZH2-H3K27me3 axis and downstream factors during both osteoclasts and osteoblasts formation and the therapeutic possibility of DZNep on bone defect healing.
METHODS
Bone marrow-derived macrophages (BMMs) cells were cultured, and their responsiveness to DZNep was evaluated by cell counting kit-8, TRAP staining assay, bone resorption assay, podosome actin belt. Bone marrow-derived mesenchymal stem cells (BMSC) were cultured and their responsiveness to DZNep was evaluated by cell counting kit-8, ALP and AR staining assay. The expression of nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), Wnt signaling pathway was determined by qPCR and western blotting. Mouse bone defect models were created, rescued by DZNep injection, and the effectiveness was evaluated by X-ray and micro-CT and histological staining.
RESULTS
Consistent with the previous study that DZNep enhances osteogenesis via Wnt family member 1(Wnt1), Wnt6, and Wnt10a, our results showed that DZNep also promotes osteoblasts differentiation and mineralization through the EZH2-H3K27me3-Wnt4 axis. Furthermore, we identified that DZNep promoted the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast formation via facilitating the phosphorylation of IKKα/β, IκB, and subsequently NF-κB nuclear translocation, which credit to the EZH2-H3K27me3-Foxc1 axis. More importantly, the enhanced osteogenesis and osteoclastogenesis result in accelerated mice bone defect healing in vivo.
CONCLUSION
DZNep targeting EZH2-H3K27me3 axis facilitated the healing of mice bone defect via simultaneously enhancing osteoclastic bone resorption and promoting osteoblastic bone formation.
Topics: Adenosine; Animals; Bone Resorption; Cell Differentiation; Epigenesis, Genetic; Mice; NF-kappa B; Osteoclasts; Osteogenesis; RANK Ligand
PubMed: 34930462
DOI: 10.1186/s13287-021-02670-6 -
Molecular Cancer Research : MCR Mar 2022Invasion of neighboring extracellular matrix (ECM) by malignant tumor cells is a hallmark of metastatic progression. This invasion can be mediated by subcellular...
UNLABELLED
Invasion of neighboring extracellular matrix (ECM) by malignant tumor cells is a hallmark of metastatic progression. This invasion can be mediated by subcellular structures known as invadopodia, the function of which depends upon soluble N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE)-mediated vesicular transport of cellular cargo. Recently, it has been shown the SNARE Syntaxin4 (Stx4) mediates trafficking of membrane type 1-matrix metalloproteinase (MT1-MMP) to invadopodia, and that Stx4 is regulated by Munc18c in this context. Here, it is observed that expression of a construct derived from the N-terminus of Stx4, which interferes with Stx4-Munc18c interaction, leads to perturbed trafficking of MT1-MMP, and reduced invadopodium-based invasion in vitro, in models of triple-negative breast cancer (TNBC). Expression of Stx4 N-terminus also led to increased survival and markedly reduced metastatic burden in multiple TNBC models in vivo. The findings are the first demonstration that disrupting Stx4-Munc18c interaction can dramatically alter metastatic progression in vivo, and suggest that this interaction warrants further investigation as a potential therapeutic target.
IMPLICATIONS
Disrupting the interaction of Syntaxin4 and Munc18c may be a useful approach to perturb trafficking of MT1-MMP and reduce metastatic potential of breast cancers.
Topics: Breast Neoplasms; Cell Line, Tumor; Extracellular Matrix; Female; Humans; Matrix Metalloproteinase 14; Neoplasm Invasiveness; Podosomes; SNARE Proteins; Triple Negative Breast Neoplasms
PubMed: 34876482
DOI: 10.1158/1541-7786.MCR-20-0527 -
Frontiers in Cell and Developmental... 2021Osteoclasts are bone resorbing cells that participate in the maintenance of bone health. Pathological increase in osteoclast activity causes bone loss, eventually...
Osteoclasts are bone resorbing cells that participate in the maintenance of bone health. Pathological increase in osteoclast activity causes bone loss, eventually resulting in osteoporosis. Actin cytoskeleton of osteoclasts organizes into a belt of podosomes, which sustains the bone resorption apparatus and is maintained by microtubules. Better understanding of the molecular mechanisms regulating osteoclast cytoskeleton is key to understand the mechanisms of bone resorption, in particular to propose new strategies against osteoporosis. We reported recently that β-tubulin isotype TUBB6 is key for cytoskeleton organization in osteoclasts and for bone resorption. Here, using an osteoclast model CRISPR/Cas9 KO for Tubb6, we show that TUBB6 controls both microtubule and actin dynamics in osteoclasts. Osteoclasts KO for Tubb6 have reduced microtubule growth speed with longer growth life time, higher levels of acetylation, and smaller EB1-caps. On the other hand, lack of TUBB6 increases podosome life time while the belt of podosomes is destabilized. Finally, we performed proteomic analyses of osteoclast microtubule-associated protein enriched fractions. This highlighted ARHGAP10 as a new microtubule-associated protein, which binding to microtubules appears to be negatively regulated by TUBB6. ARHGAP10 is a negative regulator of CDC42 activity, which participates in actin organization in osteoclasts. Our results suggest that TUBB6 plays a key role in the control of microtubule and actin cytoskeleton dynamics in osteoclasts. Moreover, by controlling ARHGAP10 association with microtubules, TUBB6 may participate in the local control of CDC42 activity to ensure efficient bone resorption.
PubMed: 34869381
DOI: 10.3389/fcell.2021.778887 -
Advanced Healthcare Materials Apr 2022A collagen-rich tumor microenvironment (TME) is associated with worse outcomes in cancer patients and contributes to drug resistance in many cancer types. In melanoma,...
A collagen-rich tumor microenvironment (TME) is associated with worse outcomes in cancer patients and contributes to drug resistance in many cancer types. In melanoma, stiff and fibrillar collagen-abundant tissue is observed after failure of therapeutic treatments with BRAF inhibitors. Increased collagen in the TME can affect properties of the extracellular matrix (ECM), including stiffness, adhesiveness, and interaction of integrins with triple helix forming nanostructures. Decoupling these biochemical and biophysical properties of the ECM can lead to a better understanding of how each of these individual properties affect melanoma cancer behavior and drug efficacy. In addition, as drug treatment can induce cancer cell phenotypic switch, cancer cell responsiveness to the TME can be dynamically changed during therapeutic treatments. To investigate cancer cell phenotype changes and the role of the cancer TME, poly(ethylene glycol) (PEG) hydrogels functionalized with collagen mimetic peptides (CMPs) is utilized, or an interpenetrating network (IPN) of type І collagen within the PEG system to culture various melanoma cell lines in the presence or absence of Vemurafenib (PLX4032) drug treatment is prepared. Additionally, the potential of using CMP functionalized PEG hydrogels, which can provide better tunability is explored, to replace the existing invadopodia assay platform based on fluorescent gelatin.
Topics: Collagen; Extracellular Matrix; Humans; Hydrogels; Melanoma; Podosomes; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Tumor Microenvironment
PubMed: 34783464
DOI: 10.1002/adhm.202101592 -
Journal of Proteomics Jan 2022Extracellular vesicles (EVs) are involved in a wide range of pathological processes and recognized as potential and novel biomarkers for oral squamous cell carcinoma...
Extracellular vesicles (EVs) are involved in a wide range of pathological processes and recognized as potential and novel biomarkers for oral squamous cell carcinoma (OSCC). Here, we describe the plasma EV proteome of rats with 4-nitroquinoline-1-oxide (4NQO)-induced OSCC or moderate dysplasia (MD), which can progress to OSCC, by tandem mass tag (TMT)-labeled mass spectrometry. The proteomic profiles suggest the differential expression of various proteins in MD and OSCC, some well-recognized pathological changes (e.g., translation, ATP metabolism, and mesenchymal transition), and some novel pathological changes (e.g., podosome, focal adhesion, and S100 binding). We re-examined the presence of traditional exosomal markers and the reported novel pan-EV markers. In summary, these results suggest potential EV biomarkers and underlying pathological changes in early OSCC as well as the presence of oral-derived EVs in plasma and the need for pan-EV markers. SIGNIFICANCE: This research suggests potential EV biomarkers and underlying pathological changes in early OSCC as well as the presence of oral-derived EVs in plasma and the need for pan-EV markers.
Topics: Animals; Carcinoma, Squamous Cell; Extracellular Vesicles; Head and Neck Neoplasms; Mouth Neoplasms; Proteome; Proteomics; Rats; Squamous Cell Carcinoma of Head and Neck
PubMed: 34775099
DOI: 10.1016/j.jprot.2021.104422