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International Journal of Molecular... Jun 2024Estrogen (17β-estradiol) deficiency post-menopause alters bone homeostasis whereby bone resorption by osteoclasts exceeds bone formation by osteoblasts, leading to...
Estrogen (17β-estradiol) deficiency post-menopause alters bone homeostasis whereby bone resorption by osteoclasts exceeds bone formation by osteoblasts, leading to osteoporosis in females. We established an in vitro model to examine the consequences of estrogen withdrawal (E2-WD) on osteoclasts derived from the mouse macrophage RAW 264.7 cell line and utilized it to investigate the mechanism behind the enhanced osteoclast activity post-menopause. We found that a greater population of osteoclasts that underwent E2-WD contained a podosome belt necessary for osteoclasts to adhere and resorb bone and possessed elevated resorptive activity compared to osteoclasts exposed to estrogen (E2) continuously. Our results show that compared to osteoclasts that received E2 continuously, those that underwent E2-WD had a faster rate of microtubule (MT) growth, reduced RhoA activation, and shorter podosome lifespan. Thus, altered podosome and MT dynamics induced by the withdrawal of estrogen supports podosome belt assembly/stability in osteoclasts, which may explain their enhanced bone resorption activity.
Topics: Animals; Osteoclasts; Mice; RAW 264.7 Cells; Estrogens; Bone Resorption; Podosomes; Microtubules; Female; rhoA GTP-Binding Protein; Estradiol; Cell Culture Techniques
PubMed: 38892322
DOI: 10.3390/ijms25116134 -
Proceedings of the National Academy of... Jun 2024Myogenesis is a multistep process that requires a spatiotemporal regulation of cell events resulting finally in myoblast fusion into multinucleated myotubes. Most major...
Myogenesis is a multistep process that requires a spatiotemporal regulation of cell events resulting finally in myoblast fusion into multinucleated myotubes. Most major insights into the mechanisms underlying fusion seem to be conserved from insects to mammals and include the formation of podosome-like protrusions (PLPs) that exert a driving force toward the founder cell. However, the machinery that governs this process remains poorly understood. In this study, we demonstrate that MTM1 is the main enzyme responsible for the production of phosphatidylinositol 5-phosphate, which in turn fuels PI5P 4-kinase α to produce a minor and functional pool of phosphatidylinositol 4,5-bisphosphate that concentrates in PLPs containing the scaffolding protein Tks5, Dynamin-2, and the fusogenic protein Myomaker. Collectively, our data reveal a functional crosstalk between a PI-phosphatase and a PI-kinase in the regulation of PLP formation.
Topics: Animals; Phosphatidylinositol Phosphates; Mice; Cell Fusion; Myoblasts; Podosomes; Protein Tyrosine Phosphatases, Non-Receptor; Muscle Development
PubMed: 38805272
DOI: 10.1073/pnas.2217971121 -
Cell Death & Disease May 2024Recruitment of fibroblasts to tumors and their activation into cancer-associated fibroblasts (CAFs) is a strategy used by tumor cells to direct extracellular matrix...
Recruitment of fibroblasts to tumors and their activation into cancer-associated fibroblasts (CAFs) is a strategy used by tumor cells to direct extracellular matrix (ECM) remodeling, invasion, and metastasis, highlighting the need to investigate the molecular mechanisms driving CAF function. Endothelin-1 (ET-1) regulates the communication between cancer and stroma and facilitates the progression of serous ovarian cancer (SOC). By binding to Endothelin A (ET) and B (ET) receptors, ET-1 enables the recruitment of β-arrestin1 (β-arr1) and the formation of signaling complexes that coordinate tumor progression. However, how ET-1 receptors might "educate" human ovarian fibroblasts (HOFs) to produce altered ECM and promote metastasis remains to be elucidated. This study identifies ET-1 as a pivotal factor in the activation of CAFs capable of proteolytic ECM remodeling and the generation of heterotypic spheroids containing cancer cells with a propensity to metastasize. An autocrine/paracrine ET-1/ETR/β-arr1 loop enhances HOF proliferation, upregulates CAF marker expression, secretes pro-inflammatory cytokines, and increases collagen contractility, and cell motility. Furthermore, ET-1 facilitates ECM remodeling by promoting the lytic activity of invadosome and activation of integrin β1. In addition, ET-1 signaling supports the formation of heterotypic HOF/SOC spheroids with enhanced ability to migrate through the mesothelial monolayer, and invade, representing metastatic units. The blockade of ETR or β-arr1 silencing prevents CAF activation, invadosome function, mesothelial clearance, and the invasive ability of heterotypic spheroids. In vivo, therapeutic inhibition of ETR using bosentan (BOS) significantly reduces the metastatic potential of combined HOFs/SOC cells, associated with enhanced apoptotic effects on tumor cells and stromal components. These findings support a model in which ET-1/β-arr1 reinforces tumor/stroma interaction through CAF activation and fosters the survival and metastatic properties of SOC cells, which could be counteracted by ETR antagonists.
Topics: Humans; Female; Ovarian Neoplasms; beta-Arrestin 1; Cancer-Associated Fibroblasts; Cell Line, Tumor; Podosomes; Endothelin-1; Neoplasm Metastasis; Receptor, Endothelin A; Signal Transduction; Extracellular Matrix; Cell Movement; Cell Proliferation; Animals; Fibroblasts; Neoplasm Invasiveness
PubMed: 38777849
DOI: 10.1038/s41419-024-06730-6 -
Cancers Apr 2024Colorectal tumorigenesis involves the development of aberrant crypt foci (ACF) or preneoplastic lesions, representing the earliest morphological lesion visible in colon...
Colorectal tumorigenesis involves the development of aberrant crypt foci (ACF) or preneoplastic lesions, representing the earliest morphological lesion visible in colon cancer. The purpose of this study was to determine changes in protein expression in carcinogen-induced ACF as they mature and transform into adenomas. Protein expression profiles of azoxymethane (AOM)-induced F344 rat colon ACF and adenomas were compared at four time points, 4 (control), 8, 16, and 24 weeks post AOM administration ( = 9/group), with time points correlating with induction and transformation events. At each time point, micro-dissected ACF and/or adenoma tissues were analyzed across multiple quantitative two-dimensional (2D-DIGE) gels using a Cy-dye labeling technique and a pooled internal standard to quantify expression changes with statistical confidence. Western blot and subsequent network pathway mapping were used to confirm and elucidate differentially expressed ( ≤ 0.05) proteins, including changes in vinculin (; = 0.007), scinderin (; = 0.02), and profilin (; = 0.01), By determining protein expression changes in ACF as they mature and transform into adenomas, a "baseline" of altered regulatory proteins associated with adenocarcinoma development in this model has been elucidated. These data will enable future studies aimed at biomarker identification and understanding the molecular biology of intestinal tumorigenesis and adenocarcinoma maturation under varying intestinal conditions.
PubMed: 38730628
DOI: 10.3390/cancers16091678 -
Cells Apr 2024Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with high mortality due to early metastatic dissemination and high chemoresistance. All these factors are...
BACKGROUND
Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with high mortality due to early metastatic dissemination and high chemoresistance. All these factors are favored by its extracellular matrix (ECM)-rich microenvironment, which is also highly hypoxic and acidic. Gemcitabine (GEM) is still the first-line therapy in PDAC. However, it is quickly deaminated to its inactive metabolite. Several GEM prodrugs have emerged to improve its cytotoxicity. Here, we analyzed how the acidic/hypoxic tumor microenvironment (TME) affects the response of PDAC cell death and invadopodia-mediated ECM proteolysis to both GEM and its C18 prodrug.
METHODS
For this, two PDAC cell lines, PANC-1 and Mia PaCa-2 were adapted to pH 6.6 or not for 1 month, grown as 3D organotypic cultures and exposed to either GEM or C18 in the presence and absence of acidosis and the hypoxia inducer, deferoxamine.
RESULTS
We found that C18 has higher cytotoxic and anti-invadopodia activity than GEM in all culture conditions and especially in acid and hypoxic environments.
CONCLUSIONS
We propose C18 as a more effective approach to conventional GEM in developing new therapeutic strategies overcoming PDAC chemoresistance.
Topics: Deoxycytidine; Gemcitabine; Humans; Tumor Microenvironment; Pancreatic Neoplasms; Cell Line, Tumor; Carcinoma, Pancreatic Ductal; Podosomes; Drug Resistance, Neoplasm; Prodrugs
PubMed: 38727266
DOI: 10.3390/cells13090730 -
European Journal of Pharmacology Jul 2024Osteoporosis is the most common bone disorder, in which an imbalance between osteoclastic bone resorption and osteoblastic bone formation disrupts bone homeostasis....
Osteoporosis is the most common bone disorder, in which an imbalance between osteoclastic bone resorption and osteoblastic bone formation disrupts bone homeostasis. Osteoporosis management using anti-osteoclastic agents is a promising strategy; however, this remains an unmet need. Sphingosine-1-phosphate (S1P) and its receptors (S1PRs) are essential for maintaining bone homeostasis. Here, we identified that Siponimod, a Food and Drug Administration-approved S1PR antagonist for the treatment of multiple sclerosis, shows promising therapeutic effects against osteoporosis by inhibiting osteoclast formation and function. We found that Siponimod inhibited osteoclast formation in a dose-dependent manner without causing cytotoxicity. Podosome belt staining and bone resorption assays indicated that Siponimod treatment impaired osteoclast function. Western blot and qPCR assays demonstrated that Siponimod suppressed the expression of osteoclast-specific markers, including C-Fos, Nftac1, and Ctsk. Mechanistically, we validated that Siponimod downregulated receptor activator of nuclear factor kappa B ligand (RANKL)-induced Mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) signaling pathways during osteoclastogenesis. Moreover, in a preclinical mouse model, Siponimod prevented ovariectomy-induced bone loss by suppressing osteoclast activity in vivo. Collectively, these results suggest that Siponimod could serve as an alternative therapeutic agent for the treatment of osteoporosis.
Topics: Animals; Mice; Osteoporosis; Osteoclasts; Drug Repositioning; Benzyl Compounds; Azetidines; Multiple Sclerosis; Female; Sphingosine 1 Phosphate Receptor Modulators; Osteogenesis; NF-kappa B; Mice, Inbred C57BL; RAW 264.7 Cells; Bone Resorption; Signal Transduction; RANK Ligand; Humans
PubMed: 38692426
DOI: 10.1016/j.ejphar.2024.176630 -
Journal of Cell Science Apr 2024Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers,...
Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14.
Topics: Female; Humans; Basigin; Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cell Movement; Extracellular Matrix; Gelatin; Lysosomal Membrane Proteins; Lysosomal-Associated Membrane Protein 1; Matrix Metalloproteinase 14; Microtubule-Associated Proteins; Monocarboxylic Acid Transporters; Muscle Proteins; Neoplasm Invasiveness; Podosomes
PubMed: 38661040
DOI: 10.1242/jcs.261608 -
BioRxiv : the Preprint Server For... Mar 2024We present single-molecule labeling and localization microscopy (SMLLM) using dye-conjugated phalloidin to achieve enhanced superresolution imaging of filamentous actin...
We present single-molecule labeling and localization microscopy (SMLLM) using dye-conjugated phalloidin to achieve enhanced superresolution imaging of filamentous actin (F-actin). We demonstrate that the intrinsic phalloidin dissociation enables SMLLM in an imaging buffer containing low concentrations of dye-conjugated phalloidin. We further show enhanced single-molecule labeling by chemically promoting phalloidin dissociation. Two benefits of phalloidin-based SMLLM are better preservation of cellular structures sensitive to mechanical and shear forces during standard sample preparation and more consistent F-actin quantification at the nanoscale. In a proof-of-concept study, we employed SMLLM to super-resolve F-actin structures in U2OS and dendritic cells (DCs) and demonstrate more consistent F-actin quantification in the cell body and structurally delicate cytoskeletal proportions, which we termed membrane fibers, of DCs compared to direct stochastic optical reconstruction microscopy (STORM). Using DC2.4 mouse dendritic cells as the model system, we show F-actin redistribution from podosomes to actin filaments and altered prevalence of F-actin-associated membrane fibers on the culture glass surface after lipopolysaccharide exposure. While our work demonstrates SMLLM for F-actin, the concept opens new possibilities for protein-specific single-molecule labeling and localization in the same step using commercially available reagents.
PubMed: 38496456
DOI: 10.1101/2024.03.04.583337 -
Biochemical and Biophysical Research... Apr 2024Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the...
Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the stiffness of bones: regular calcified bone (bone that is resorbable by osteoclasts) and un-calcified osteoid bone (bone that is un-resorbable by osteoclasts). The osteolytic cytokine RANKL promotes osteoclast differentiation; however, the roles of the physical interactions of osteoclasts with calcified and un-calcified bone at the sealing zones and the subsequent cellular signaling remain unclear. In this study, we investigated podosomes, actin-rich adhesion structures (actin-ring) in the sealing zone that participates in sensing hard stiffness with collagen in the physical environment during osteoclast differentiation. RANKL-induced osteoclast differentiation induction was promoted when Raw264.7 cells were cultured on collagen-coated plastic dishes but not on non-coated plastic dishes, which was associated with the increased expression of podosome-related genes and Src. In contrast, when cells were cultured on collagen gel, expression of podosome-related genes and Src were not upregulated. The induction of podosome-related genes and Src requires hard stiffness with RGD-containing substratum and integrin-mediated F-actin polymerization. These results indicate that osteoclasts sense both the RGD sequence and stiffness of calcified collagen through their podosome components regulating osteoclast differentiation via the c-Src pathway.
Topics: Humans; Osteoclasts; Podosomes; Actins; Cell Differentiation; Bone Resorption; CSK Tyrosine-Protein Kinase; Collagen; Oligopeptides
PubMed: 38402724
DOI: 10.1016/j.bbrc.2024.149636 -
Cell Reports Feb 2024The acidic metabolic byproducts within the tumor microenvironment (TME) hinder T cell effector functions. However, their effects on T cell infiltration remain largely...
The acidic metabolic byproducts within the tumor microenvironment (TME) hinder T cell effector functions. However, their effects on T cell infiltration remain largely unexplored. Leveraging the comprehensive The Cancer Genome Atlas dataset, we pinpoint 16 genes that correlate with extracellular acidification and establish a metric known as the "tumor acidity (TuAci) score" for individual patients. We consistently observe a negative association between the TuAci score and T lymphocyte score (T score) across various human cancer types. Mechanistically, extracellular acidification significantly impedes T cell motility by suppressing podosome formation. This phenomenon can be attributed to the reduced expression of methyltransferase-like 3 (METTL3) and the modification of RNA N-methyladenosine (mA), resulting in a subsequent decrease in the expression of integrin β1 (ITGB1). Importantly, enforced ITGB1 expression leads to enhanced T cell infiltration and improved antitumor activity. Our study suggests that modulating METTL3 activity or boosting ITGB1 expression could augment T cell infiltration within the acidic TME, thereby improving the efficacy of cell therapy.
Topics: Humans; Cell- and Tissue-Based Therapy; Hydrogen-Ion Concentration; Integrin beta1; Methyltransferases; Neoplasms; T-Lymphocytes; Tumor Microenvironment
PubMed: 38367240
DOI: 10.1016/j.celrep.2024.113796