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Acta Biomaterialia Sep 2023In vivo bone remodeling is promoted by the balance between osteoclast and osteoblast activity. Conventional research on bone regeneration has mainly focused on...
In vivo bone remodeling is promoted by the balance between osteoclast and osteoblast activity. Conventional research on bone regeneration has mainly focused on increasing osteoblast activity, with limited studies on the effects of scaffold topography on cell differentiation. Here, we examined the effect of microgroove-patterned substrate with spacings ranging from 1 to 10 μm on the differentiation of rat bone marrow-derived osteoclast precursors. Tartrate-resistant acid phosphatase (TRAP) staining and relative gene expression quantification showed that osteoclast differentiation was enhanced in substrate with 1 µm microgroove spacing compared with that in the other groups. Additionally, the ratio of podosome maturation stages in substrate with 1 μm microgroove spacing exhibited a distinct pattern, which was characterized by an increase in the ratio of belts and rings and a decrease in that of clusters. However, myosin II abolished the effects of topography on osteoclast differentiation. Overall, these showed that the reduction of myosin II tension in the podosome core by an integrin vertical vector increased podosome stability and promoted osteoclast differentiation in substrates with 1 μm microgroove spacing, including that microgroove design plays an important role in scaffolds for bone regeneration. STATEMENT OF SIGNIFICANCE: Reduction of myosin II tension in the podosome core, facilitated by an integrin vertical vector, resulted in an enhanced osteoclast differentiation, concomitant with an increase in podosome stability within 1-μm-spaced microgrooves. These findings are anticipated to serve as valuable indicators for the regulation of osteoclast differentiation through the manipulation of biomaterial surface topography in tissue engineering. Furthermore, this study contributes to the lucidation of the underlying mechanisms governing cellular differentiation by providing insights into the impact of the microtopographical environment.
Topics: Rats; Animals; Osteoclasts; Cell Differentiation; Osteoblasts; Bone Remodeling; Integrins
PubMed: 37392936
DOI: 10.1016/j.actbio.2023.06.033 -
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 -
The Journal of Investigative Dermatology Aug 2023Signaling through the HGF receptor/Met in skin-resident Langerhans cells (LCs) and dermal dendritic cells (DCs) is essential for their emigration toward draining lymph...
Signaling through the HGF receptor/Met in skin-resident Langerhans cells (LCs) and dermal dendritic cells (DCs) is essential for their emigration toward draining lymph nodes upon inflammation-induced activation. In this study, we addressed the role of Met signaling in distinct steps of LC/dermal DC emigration from the skin by employing a conditionally Met-deficient mouse model (Met). We found that Met deficiency severely impaired podosome formation in DCs and concomitantly decreased the proteolytic degradation of gelatin. Accordingly, Met-deficient LCs failed to efficiently cross the extracellular matrix-rich basement membrane between the epidermis and the dermis. We further observed that HGF-dependent Met activation reduced the adhesion of bone marrow-derived LCs to various extracellular matrix factors and enhanced the motility of DCs in three-dimensional collagen matrices, which was not the case for Met-deficient LCs/DCs. We found no impact of Met signaling on the integrin-independent amoeboid migration of DCs in response to the CCR7 ligand CCL19. Collectively, our data show that the Met-signaling pathway regulates the migratory properties of DC in HGF-dependent and HGF-independent manners.
Topics: Mice; Animals; Podosomes; Cell Movement; Skin; Langerhans Cells; Signal Transduction; Dendritic Cells; Lymph Nodes
PubMed: 36813160
DOI: 10.1016/j.jid.2022.12.025 -
Bulletin of Mathematical Biology Feb 2024One of the most crucial and lethal characteristics of solid tumors is represented by the increased ability of cancer cells to migrate and invade other organs during the...
One of the most crucial and lethal characteristics of solid tumors is represented by the increased ability of cancer cells to migrate and invade other organs during the so-called metastatic spread. This is allowed thanks to the production of matrix metalloproteinases (MMPs), enzymes capable of degrading a type of collagen abundant in the basal membrane separating the epithelial tissue from the connective one. In this work, we employ a synergistic experimental and mathematical modelling approach to explore the invasion process of tumor cells. A mathematical model composed of reaction-diffusion equations describing the evolution of the tumor cells density on a gelatin substrate, MMPs enzymes concentration and the degradation of the gelatin is proposed. This is completed with a calibration strategy. We perform a sensitivity analysis and explore a parameter estimation technique both on synthetic and experimental data in order to find the optimal parameters that describe the in vitro experiments. A comparison between numerical and experimental solutions ends the work.
Topics: Humans; Podosomes; Gelatin; Extracellular Matrix; Models, Biological; Mathematical Concepts; Matrix Metalloproteinases; Neoplasm Invasiveness
PubMed: 38347328
DOI: 10.1007/s11538-024-01260-w -
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 -
The Science of the Total Environment Mar 2024Since European regulators restricted the use of bacteriocidic triclosan (TCS), alternatives for TCS are emerging. Recently, TCS has been shown to reprogram immune...
Since European regulators restricted the use of bacteriocidic triclosan (TCS), alternatives for TCS are emerging. Recently, TCS has been shown to reprogram immune metabolism, trigger the NLRP3 inflammasome, and subsequently the release of IL-1β in human macrophages, but data on substitutes is scarce. Hence, we aimed to examine the effects of TCS compared to its alternatives at the molecular level in human macrophages. LPS-stimulated THP-1 macrophages were exposed to TCS or its substitutes, including benzalkonium chloride, benzethonium chloride, chloroxylenol, chlorhexidine (CHX) and cetylpyridinium chloride, with the inhibitory concentration (IC-value) of cell viability to decipher their mode of action. TCS induced the release of the pro-inflammatory cytokine TNF and high level of IL-1β, suggesting the activation of the NLRP3-inflammasome, which was confirmed by non-apparent IL-1β under the NLRP3-inhibitor MCC950 treatment d. While IL-6 release was reduced in all treatments, the alternative CHX completely abolished the release of all investigated cytokines. To unravel the underlying molecular mechanisms, we used untargeted LC-MS/MS-based proteomics. TCS and CHX showed the strongest cellular response at the protein and signalling pathway level, whereby pathways related to metabolism, translation, cellular stress and migration were mainly affected but to different proposed modes of action. TCS inhibited mitochondrial electron transfer and affected phagocytosis. In contrast, in CHX-treated cells, the translation was arrested due to stress conditions, resulting in the formation of stress granules. Mitochondrial (e.g. ATP5F1D, ATP5PB, UQCRQ) and ribosomal (e.g. RPL10, RPL35, RPS23) proteins were revealed as putative key drivers. Furthermore, we have demonstrated the formation of podosomes by CHX, potentially involved in ECM degradation. Our results exhibit modulation of the immune response in macrophages by TCS and its substitutes and illuminated underlying molecular effects. These results illustrate critical processes involved in the modulation of macrophages' immune response by TCS and its alternatives, providing information essential for hazard assessment.
Topics: Humans; NLR Family, Pyrin Domain-Containing 3 Protein; Inflammasomes; Triclosan; Chlorhexidine; Chromatography, Liquid; Tandem Mass Spectrometry; Macrophages; Interleukin-1beta; Cytokines; Immunity
PubMed: 38159774
DOI: 10.1016/j.scitotenv.2023.169650 -
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 -
Phytomedicine : International Journal... Jul 2024Osteoclast plays an important role in maintaining the balance between bone anabolism and bone catabolism. The abnormality of osteoclast is closely related to osteolytic...
BACKGROUND
Osteoclast plays an important role in maintaining the balance between bone anabolism and bone catabolism. The abnormality of osteoclast is closely related to osteolytic bone diseases such as osteoporosis, rheumatoid arthritis and tumor bone metastasis.
PURPOSE
We aim to search for natural compound that may suppress osteoclast formation and function.
STUDY DESIGN
In this study, we assessed the impact of Dauricine (Dau) on the formation and function of osteoclasts in vitro, as well as its potential in preventing bone loss in an ovariectomy mouse model in vivo.
METHODS
Multiple in vitro experiments were carried out, including osteoclastogenesis, podosomal belt formation, bone resorption assay, RNA-sequencing, real-time quantitative PCR, ROS level detection, surface plasmon resonance assay, luciferase assay and western blot. To verify the effect in vivo, an ovariectomized mouse model (OVX model) was constructed, and bone parameters were measured using micro-CT and histology. Furthermore, metabolomics analysis was performed on blood serum samples from the OVX model.
RESULTS
In vitro experiments demonstrated that Dau inhibits RANKL-induced osteoclastogenesis, podosomal belt formation, and bone resorption function. RNA-sequencing results revealed that Dau significantly suppresses genes related to osteoclast. Functional enrichment analysis indicated that Dau's inhibition of osteoclasts may be associated with NF-κB signaling pathway and reactive oxygen metabolism pathway. Molecular docking, surface plasmon resonance assay and western blot analysis further confirmed that Dau inhibits RANKL-induced osteoclastogenesis by modulating the ROS/NF-κB/NFATc1 pathway. Moreover, administration of Dau to OVX-induced mice validated its efficacy in treating bone loss disease.
CONCLUSION
Dau prevents OVX-induced bone loss by inhibiting osteoclast activity and bone resorption, potentially offering a new approach for preventing and treating metabolic bone diseases such as osteoporosis. This study provides innovative insights into the inhibitory effects of Dau in an in vivo OVX model and elucidates the underlying mechanism.
Topics: Animals; Ovariectomy; Benzylisoquinolines; Female; RANK Ligand; Mice; NF-kappa B; Reactive Oxygen Species; Osteogenesis; Osteoclasts; NFATC Transcription Factors; Disease Models, Animal; Bone Resorption; Mice, Inbred C57BL; RAW 264.7 Cells; Osteoporosis; Humans; Tetrahydroisoquinolines
PubMed: 38579642
DOI: 10.1016/j.phymed.2024.155559 -
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 -
Journal of Immunology Research 2023T-cell Immunoglobulin and Mucin Domain 3 (TIM-3) is an immune checkpoint receptor known to regulate T-cell activation and has been targeted for immunotherapy in cancer...
T-cell Immunoglobulin and Mucin Domain 3 (TIM-3) is an immune checkpoint receptor known to regulate T-cell activation and has been targeted for immunotherapy in cancer and other diseases. However, its expression and function in other cell types, such as macrophages, are poorly understood. This study investigated TIM-3 expression in human macrophages polarized to M1 (stimulated with IFN- and LPS) and M2 (stimulated with IL-4 and IL-13) phenotypes using an in vitro model. Our results show that M1 macrophages have a lower frequency of TIM-3+ cells compared to M2 macrophages at 48 and 72 hr poststimulation. Additionally, we observed differential levels of soluble ADAM 10, an enzyme responsible for TIM-3 release, in the supernatants of M1 and M2 macrophages at 72 hr. We also found that the TIM-3 intracellular tail might associate with lymphocyte-specific protein 1 (LSP-1), a protein implicated in cell motility and podosome formation. These findings enhance our understanding of TIM-3 function in myeloid cells such as macrophages and may inform the development of immunotherapies with reduced immune-related adverse effects.
Topics: Humans; Hepatitis A Virus Cellular Receptor 2; Macrophages; Microfilament Proteins
PubMed: 37928435
DOI: 10.1155/2023/3577334