Asthmatic Eosinophils Alter the Gene Expression of Extracellular Matrix Proteins in Airway Smooth Muscle Cells and Pulmonary Fibroblasts

The impaired production of extracellular matrix (ECM) proteins by airway smooth muscle cells (ASMC) and pulmonary fibroblasts (PF) is a part of airway remodeling in asthma. This process might be influenced by eosinophils that migrate to the airway and abundantly secrete various cytokines, including TGF-β. We aimed to investigate the effect of asthmatic eosinophils on the gene expression of ECM proteins in ASMC and PF. A total of 34 study subjects were recruited: 14 with allergic asthma (AA), 9 with severe non-allergic eosinophilic asthma (SNEA), and 11 healthy subjects (HS). All AA patients underwent bronchial allergen challenge with D. pteronyssinus. The peripheral blood eosinophils were isolated using high-density centrifugation and magnetic separation. The individual cell cultures were made using hTERT ASMC and MRC-5 cell lines and the subjects’ eosinophils. The gene expression of ECM and the TGF-β signaling pathway was analyzed using qRT-PCR. We found that asthmatic eosinophils significantly promoted collagen I, fibronectin, versican, tenascin C, decorin, vitronectin, periostin, vimentin, MMP-9, ADAM33, TIMP-1, and TIMP-2 gene expression in ASMC and collagen I, collagen III, fibronectin, elastin, decorin, MMP-2, and TIMP-2 gene expression in PF compared with the HS eosinophil effect. The asthmatic eosinophils significantly increased the gene expression of several canonical and non-canonical TGF-β signaling pathway components in ASMC and PF compared with the HS eosinophil effect. The allergen-activated AA and SNEA eosinophils had a greater effect on these changes. In conclusion, asthmatic eosinophils, especially SNEA and allergen-activated eosinophils, imbalanced the gene expression of ECM proteins and their degradation-regulating proteins. These changes were associated with increased gene expression of TGF-β signaling pathway molecules in ASMC and PF.     

Effects of Tenascin C on the Integrity of Extracellular Matrix and Skin Aging

Tenascin C (TNC) is an element of the extracellular matrix (ECM) of various tissues, including the skin, and is involved in modulating ECM integrity and cell physiology. Although skin aging is apparently associated with changes in the ECM, little is known about the role of TNC in skin aging. In this study, we found that the Tnc mRNA level was significantly reduced in the skin tissues of aged mice compared with young mice, consistent with reduced TNC protein expression in aged human skin. TNC-large (TNC-L; 330-kDa) and -small (TNC-S; 240-kDa) polypeptides were observed in conditional media from primary dermal fibroblasts. Both recombinant TNC polypeptides, corresponding to TNC-L and TNC-S, increased the expression of type I collagen and reduced the expression of matrix metalloproteinase-1 in fibroblasts. Treatment of fibroblasts with a recombinant TNC polypeptide, corresponding to TNC-L, induced phosphorylation of SMAD2 and SMAD3. TNC increased the level of transforming growth factor-β1 (TGF-β1) mRNA and upregulated the expression of type I collagen by activating the TGF-β signaling pathway. In addition, TNC also promoted the expression of type I collagen in fibroblasts embedded in a three-dimensional collagen matrix. Our findings suggest that TNC contributes to the integrity of ECM in young skin and to prevention of skin aging.     

Integrin β1 Promotes Pancreatic Tumor Growth by Upregulating Kindlin-2 and TGF-β Receptor-2

The tumor microenvironment plays a critical role in defining the growth and malignancy of solid tumors. Extracellular matrix (ECM) proteins such as collagen, vitronectin, and fibronectin are major components of the tumor microenvironment. Tumor growth-promoting reciprocal interaction between ECM and cytoplasmic proteins is regulated by the cell surface receptors called integrins. This study investigated the mechanism by which integrin β1 promotes pancreatic tumor growth. In MIA PaCa-2 pancreatic cancer cell line, the loss of integrin β1 protein reduced the ability of cells to proliferate in a 3D matrix and compromised the ability to form a focal adhesion complex. Decreased expression of integrin α5 was observed in KO cells, which resulted in impaired cell spreading and adhesion on vitronectin and fibronectin. Reduced expression of the integrin-associated protein, kindlin-2 was also recorded. The downregulation of kindlin-2 decreased the phosphorylation of Smad2/3 by reducing the expression of TGF-β receptor 2. These results unravel a new mechanism of integrin β1 in tumor growth by modifying the expression of kindlin-2 and TGF-β receptor 2 signaling.     

MMP9 Differentially Regulates Proteins Involved in Actin Polymerization and Cell Migration during TGF-β-Induced EMT in the Lens

Fibrotic cataracts have been attributed to transforming growth factor-beta (TGF-β)-induced epithelial-to-mesenchymal transition (EMT). Using mouse knockout (KO) models, our laboratory has identified MMP9 as a crucial protein in the TGF-β-induced EMT process. In this study, we further revealed an absence of alpha-smooth muscle actin (αSMA) and filamentous-actin (F-actin) stress fibers in MMP9KO mouse lens epithelial cell explants (LECs). Expression analysis using NanoString revealed no marked differences in αSMA (ACTA2) and beta-actin (β-actin) (ACTB) mRNA between the lenses of TGF-β-overexpressing (TGF-βtg) mice and TGF-βtg mice on a MMP9KO background. We subsequently conducted a protein array that revealed differential regulation of proteins known to be involved in actin polymerization and cell migration in TGF-β-treated MMP9KO mouse LECs when compared to untreated controls. Immunofluorescence analyses using rat LECs and the novel MMP9-specific inhibitor, JNJ0966, revealed similar differential regulation of cortactin, FAK, LIMK1 and MLC2 as observed in the array. Finally, a reduction in the nuclear localization of MRTF-A, a master regulator of cytoskeletal remodeling during EMT, was observed in rat LECs co-treated with JNJ0966 and TGF-β. In conclusion, MMP9 deficiency results in differential regulation of proteins involved in actin polymerization and cell migration, and this in turn prevents TGF-β-induced EMT in the lens.     

Decorin—An Antagonist of TGF-β in Astrocytes of the Optic Nerve

During the pathogenesis of glaucoma, optic nerve (ON) axons become continuously damaged at the optic nerve head (ONH). This often is associated with reactive astrocytes and increased transforming growth factor (TGF-β) 2 levels. In this study we tested the hypothesis if the presence or absence of decorin (DCN), a small leucine-rich proteoglycan and a natural inhibitor of several members of the TGF family, would affect the expression of the TGF-βs and connective tissue growth factor (CTGF/CCN2) in human ONH astrocytes and murine ON astrocytes. We found that DCN is present in the mouse ON and is expressed by human ONH and murine ON astrocytes. DCN expression and synthesis was significantly reduced after 24 h treatment with 3 nM CTGF/CCN2, while treatment with 4 pM TGF-β2 only reduced expression of DCN significantly. Conversely, DCN treatment significantly reduced the expression of TGF-β1, TGF-β2 and CTGF/CCN2 vis-a-vis untreated controls. Furthermore, DCN treatment significantly reduced expression of fibronectin (FN) and collagen IV (COL IV). Notably, combined treatment with DCN and triciribine, a small molecule inhibitor of protein kinase B (AKT), attenuated effects of DCN on CTGF/CCN2, TGF-β1, and TGF-β2 mRNA expression. We conclude (1) that DCN is an important regulator of TGF-β and CTGF/CCN2 expression in astrocytes of the ON and ONH, (2) that DCN thereby regulates the expression of extracellular matrix (ECM) components and (3) that DCN executes its negative regulatory effects on TGF-β and CTGF/CCN2 via the pAKT/AKT signaling pathway in ON astrocytes.     

Synthesis of Reactive Sulfur Species in Cultured Vascular Endothelial Cells after Exposure to TGF-β1: Induction of Cystathionine γ-Lyase and Cystathionine β-Synthase Expression Mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 Pathways

Transforming growth factor-β₁ (TGF-β₁) occurs at high levels at damage sites of vascular endothelial cell layers and regulates the functions of vascular endothelial cells. Reactive sulfur species (RSS), such as cysteine persulfide, glutathione persulfide, and hydrogen persulfide, are cytoprotective factors against electrophiles such as reactive oxygen species and heavy metals. Previously, we reported that sodium trisulfide, a sulfane sulfur donor, promotes vascular endothelial cell proliferation. The objective of the present study was to clarify the regulation and significance of RSS synthesis in vascular endothelial cells after exposure to TGF-β₁. Bovine aortic endothelial cells in a culture system were treated with TGF-β₁ to assess the expression of intracellular RSS, the effect of RSS on cell proliferation in the presence of TGF-β₁, induction of RSS-producing enzymes by TGF-β₁, and intracellular signal pathways that mediate this induction. The results suggest that TGF-β₁ increased intracellular RSS levels to modulate its inhibitory effect on proliferation. The increased production of RSS, probably high-molecular-mass RSS, was due to the induction of cystathionine γ-lyase and cystathionine β-synthase, which are RSS-producing enzymes, and the induction was mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 pathways in vascular endothelial cells. TGF-β₁ regulates vascular endothelial cell functions such as proliferation and fibrinolytic activity; intracellular high-molecular-mass RSS, which are increased by TGF-β₁, may modulate the regulation activity in vascular endothelial cells.     

Heightened Crescentic Glomerulonephritis in Immune Challenged 129sv Mice Is TGF-β/Smad3 Dependent

The 129sv mouse strain is particularly sensitive to experimental immune-mediated nephritis. Previous studies have indicated that transforming growth factor-β (TGF-β) plays a critical role in both immune modulation and tissue fibrogenesis in various diseases and that its biological activities are exerted via the SMAD family. In this study, we aimed to determine whether TGF-β/SMAD signaling is essential for the development of immune-mediated nephritis in 129sv mice. Relative to C57BL/6J control mice with anti-glomeruli basement membrane (GBM) nephritis, 129sv mice with anti-GBM nephritis exhibited increased renal collagen deposition. Additionally, higher mRNA levels of pro-collagen and collagen IV, higher serum levels of active and total TGF-β1, and increased TGF-β1, TGF-βIIR, and phosphorylated SMAD expression were detected in these mice. Deletion of Smad3 in 129sv mice ameliorated anti-GBM induced nephritis, including crescentic glomerulonephritis. Collectively, these findings indicate that the heightened experimental nephritis and fibrotic disease in the 129sv strain of mice are regulated by SMAD3, which could be a potential therapeutic target for immune-mediated nephritis.     

Metformin Prevents Renal Fibrosis in Mice with Unilateral Ureteral Obstruction and Inhibits Ang II-Induced ECM Production in Renal Fibroblasts

Renal fibrosis is the final common pathway of chronic kidney disease (CKD), and no effective medication is available clinically for managing its progression. Metformin was initially developed as an anti-diabetic drug and recently gained attention for its potential in the treatment of other diseases. In this study, we investigated its effects on renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO) in vivo and in angiotensin II (Ang II)–treated renal fibroblast NRK-49F cells in vitro. Our data showed that UUO induced renal fibrosis and combined with the activation of ERK signaling, the upregulation of fibronectin, collagen I, and transforming growth factor-β (TGF-β). The administration of metformin inhibited the activation of ERK signaling and attenuated the production of extracellular matrix (ECM) proteins and collagen deposition in the obstructed kidneys. In cultured renal fibroblasts, Ang II increased the expression of fibronectin and collagen I and also activated ERK signaling and TGF-β in a time-dependent manner. Pretreatment of the cells with metformin blocked Ang II–induced ERK signaling activation and ECM overproduction. Our results show that metformin prevents renal fibrosis, possibly through the inhibition of ERK signaling, and may be a novel strategy for the treatment of renal fibrosis.     

Astragalus membranaceus Inhibits Peritoneal Fibrosis via Monocyte Chemoattractant Protein (MCP)-1 and the Transforming Growth Factor-β1 (TGF-β1) Pathway in Rats Submitted to Peritoneal Dialysis

Inflammation and transforming growth factor-β1 (TGF-β1) contribute to the development of peritoneal fibrosis (PF), which is associated with peritoneal dialysis (PD). Astragalus membranaceus (Astragalus) has anti-inflammatory and anti-fibrotic effects in many diseases. The goal of this study was to determine the anti-fibrotic effects of Astragalus on the PF response to PD. A rat model of PD was induced using standard PD fluid, and PF was verified by HE and Masson’s staining, as well as through the expression of fibroblast surface protein (FSP) and collagen III. The expression levels of monocyte chemoattractant protein (MCP)-1, F4/80 (macrophage/monocyte marker in rat), TGF-β1 and the downstream proteins phospho-SMAD 2/3 in dialyzed peritoneal tissue treated with or without Astragalus was evaluated using immunohistochemistry analysis. Overall correlations between MCP-1 and TGF-β1 staining were analyzed using both the Spearman and Pearson methods. The results showed that Astragalus could inhibit the recruitment and activation of monocytes/macrophages, thereby reducing the production of TGF-β1 in the dialyzed peritoneal membrane. PF was also significantly decreased following treatment with Astragalus. MCP-1 expression had a strong positive correlation with TGF-β1 sensitivity, suggesting that the anti-fibrotic function of Astragalus was mediated by MCP-1 and the TGF-β1 pathway. Our results indicate that Astragalus could be a useful agent against PD-induced PF.     

Nuclear Respiratory Factor-1, a Novel SMAD4 Binding Protein,Represses TGF-β/SMAD4 Signaling by Functioning as a Transcriptional Cofactor

SMAD4, a key regulator of transforming growth factor-β (TGF-β) signaling, plays a major role in cell growth, migration, and apoptosis. In particular, TGF-β/SMAD induces growth arrest, and SMAD4 induces the expression of target genes such as p21WAF1 and p15INK4b through its interaction with several cofactors. Thus, inactivating mutations or the homozygous deletion of SMAD4 could be related to tumorigenesis or malignancy progression. However, in some cancer types, SMAD4 is neither mutated nor deleted. In the current study, we demonstrate that TGF-β signaling with a preserved SMAD4 function can contribute to cancer through associations with negative pathway regulators. We found that nuclear respiratory factor-1 (NRF1) is a novel interaction SMAD4 partner that inhibits TGF-β/SMAD4-induced p15INK4b mRNA expression by binding to SMAD4. Furthermore, we confirmed that NRF1 directly binds to the core region of the SMAD4 promoter, thereby decreasing SMAD4 mRNA expression. On the whole, our data suggest that NRF1 is a negative regulator of SMAD4 and can interfere with TGF-β/SMAD-induced tumor suppression. Our findings provide a novel perception into the molecular basis of TGF-β/SMAD4-signaling suppression in tumorigenesis.     

Hypoxia Differently Affects TGF-β2-Induced Epithelial Mesenchymal Transitions in the 2D and 3D Culture of the Human Retinal Pigment Epithelium Cells

The hypoxia associated with the transforming growth factor-β2 (TGF-β2)-induced epithelial mesenchymal transition (EMT) of human retinal pigment epithelium (HRPE) cells is well recognized as the essential underlying mechanism responsible for the development of proliferative retinal diseases. In vitro, three-dimensional (3D) models associated with spontaneous O₂ gradients can be used to recapitulate the pathological levels of hypoxia to study the effect of hypoxia on the TGF-β2-induced EMT of HRPE cells in detail, we used two-dimensional-(2D) and 3D-cultured HRPE cells. TGF-β2 and hypoxia significantly and synergistically increased the barrier function of the 2D HRPE monolayers, as evidenced by TEER measurements, the downsizing and stiffening of the 3D HRPE spheroids and the mRNA expression of most of the ECM proteins. A real-time metabolic analysis indicated that TGF-β2 caused a decrease in the maximal capacity of mitochondrial oxidative phosphorylation in the 2D HRPE cells, whereas, in the case of 3D HRPE spheroids, TGF-β2 increased proton leakage. The findings reported herein indicate that the TGF-β2-induced EMT of both the 2D and 3D cultured HRPE cells were greatly modified by hypoxia, but during these EMT processes, the metabolic plasticity was different between 2D and 3D HRPE cells, suggesting that the mechanisms responsible for the EMT of the HRPE cells may be variable during their spatial spreading.     

SMA-10 Is a Non-Canonical Member of the TGF-β Sma/Mab Pathway and Immunity Regulator via the DAF-2 Insulin Receptor in Caenorhabditis elegans

Transforming growth factor β (TGF-β) signalling pathways are highly conserved across metazoa and play essential roles not only during development but also in adult tissue maintenance. Alterations of these pathways usually result in a plethora of pathologies. In the nematode Caenorhabditis elegans, the TGF-β Sma/Mab (small/male abnormal) pathway regulates various worm phenotypes such as body size, immune response, ageing, matricide and reproductive span. SMA-10 has been described as a positive modulator of worm body size through the TGF-β Sma/Mab pathway. To better understand if SMA-10 is a core component of the pathway, we use gene epistatic analysis to assess the contribution of SMA-10 to various phenotypes regulated by TGF-β Sma/Mab. We confirm that SMA-10 controls body size and find that it also affects the matricide and reproductive span of the nematodes. However, neither male tail formation (previously reported) nor ageing appeared altered. Lastly, although null sma-10 worms are more susceptible to Pseudomonas aeruginosa infections than wild-types, this response does not depend on TGF-β Sma/Mab but on the insulin receptor DAF-2. We also show that the expression of sma-10 in either hypodermis or intestine fully rescues the wild-type immune response. Our results contribute to understanding the role of SMA-10 as a context-dependent component of TGF-β Sma/Mab, and reveal a function of SMA-10 in immunity in association to the Insulin/insulin-like growth factor signalling (IIS) pathway.     

Oxidative Stress Enhances the TGF-β2-RhoA-MRTF-A/B Axis in Cells Entering Endothelial-Mesenchymal Transition

Around 45% of deaths in the EU and the US are due to fibrotic diseases. Although myofibroblasts are detected in various fibrotic tissues, they are mostly transdifferentiated from endothelial cells during the endothelial-mesenchymal transition (EndMT) induced by tumor growth factor-beta (TGF-β) family members. Growing evidence indicates that oxidative stress might enhance the sensitivity and the effects of TGF-β stimulation; however, the molecular mechanisms involved in the coordination of oxidative stress and TGF-β inductions remain poorly understood. Our findings indicate for the first time that oxidative stress enhances mesenchymal trans-differentiation of human microvascular endothelial cells (HMEC-1 cells) and that the oxidative stress-dependent TGF-β2-RhoA/Rac1-MRTF-A axis is critical for the induction of later stages of EndMT. This additive effect was manifested in TGF-β1-stimulated and Snail-overexpressed cells, where it caused higher cell elongation and faster migration on collagen I layers. Additionally, Western blot assay indicated the presence of alterations in cell contraction and EndMT markers. We conclude that complex anti-fibrotic therapies based on the inhibition of MRTF activities and oxidative stress might be an attractive target for fibrosis treatment.     

WNT/β-Catenin Signaling Promotes TGF-β-Mediated Activation of Human Cardiac Fibroblasts by Enhancing IL-11 Production

Cardiac fibrosis is a pathological process associated with the development of heart failure. TGF-β and WNT signaling have been implicated in pathogenesis of cardiac fibrosis, however, little is known about molecular cross-talk between these two pathways. The aim of this study was to examine the effect of exogenous canonical WNT3a and non-canonical WNT5a in TGF-β-activated human cardiac fibroblasts. We found that WNT3a and TGF-β induced a β-catenin-dependent response, whereas WNT5a prompted AP-1 activity. TGF-β triggered profibrotic signatures in cardiac fibroblasts, and co-stimulation with WNT3a or co-activation of the β-catenin pathway with the GSK3β inhibitor CHIR99021 enhanced collagen I and fibronectin production and development of active contractile stress fibers. In the absence of TGF-β, neither WNT3a nor CHIR99021 exerted profibrotic responses. On a molecular level, in TGF-β-activated fibroblasts, WNT3a enhanced phosphorylation of TAK1 and production and secretion of IL-11 but showed no effect on the Smad pathway. Neutralization of IL-11 activity with the blocking anti-IL-11 antibody effectively reduced the profibrotic response of cardiac fibroblasts activated with TGF-β and WNT3a. In contrast to canonical WNT3a, co-activation with non-canonical WNT5a suppressed TGF-β-induced production of collagen I. In conclusion, WNT/β-catenin signaling promotes TGF-β-mediated fibroblast-to-myofibroblast transition by enhancing IL-11 production. Thus, the uncovered mechanism broadens our knowledge on a molecular basis of cardiac fibrogenesis and defines novel therapeutic targets for fibrotic heart diseases.     

CCN2 Increases TGF-β Receptor Type II Expression in Vascular Smooth Muscle Cells: Essential Role of CCN2 in the TGF-β Pathway Regulation

The cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a mediator of the fibrotic responses induced by other factors including the transforming growth factor β (TGF-β). However, several studies have defined a direct role of CCN2 acting as a growth factor inducing oxidative and proinflammatory responses. The presence of CCN2 and TGF-β together in the cellular context has been described as a requisite to induce a persistent fibrotic response, but the precise mechanisms implicated in this relation are not described yet. Considering the main role of TGF-β receptors (TβR) in the TGF-β pathway activation, our aim was to investigate the effects of CCN2 in the regulation of TβRI and TβRII levels in vascular smooth muscle cells (VSMCs). While no differences were observed in TβRI levels, an increase in TβRII expression at both gene and protein level were found 48 h after stimulation with the C-terminal fragment of CCN2 (CCN2(IV)). Cell pretreatment with a TβRI inhibitor did not modify TβRII increment induced by CCN2(VI), demonstrating a TGF-β-independent response. Secondly, CCN2(IV) rapidly activated the SMAD pathway in VSMCs, this being crucial in the upregulation of TβRII since the preincubation with an SMAD3 inhibitor prevented it. Similarly, pretreatment with the epidermal growth factor receptor (EGFR) inhibitor erlotinib abolished TβRII upregulation, indicating the participation of this receptor in the observed responses. Our findings suggest a direct role of CCN2 maintaining the TGF-β pathway activation by increasing TβRII expression in an EGFR-SMAD dependent manner activation.     

The Wnt11 Signaling Pathway in Potential Cellular EMT and Osteochondral Differentiation Progression in Nephrolithiasis Formation

The molecular events leading to nephrolithiasis are extremely complex. Previous studies demonstrated that calcium and transforming growth factor-β1 (TGF-β1) may participate in the pathogenesis of stone formation, but the explicit mechanism has not been defined. Using a self-created genetic hypercalciuric stone-forming (GHS) rat model, we observed that the increased level of serous/uric TGF-β1 and elevated intracellular calcium in primary renal tubular epithelial cells (PRECs) was associated with nephrolithiasis progression in vivo. In the setting of high calcium plus high TGF-β1 in vitro, PRECs showed great potential epithelial to mesenchymal transition (EMT) progression and osteochondral differentiation properties, representing the multifarious increased mesenchymal and osteochondral phenotypes (Zeb1, Snail1, Col2A1, OPN, Sox9, Runx2) and decreased epithelial phenotypes (E-cadherin, CK19) bythe detection of mRNAs and corresponding proteins. Moreover, TGF-β-dependent Wnt11 knockdown and L-type Ca²⁺ channel blocker could greatly reverse EMT progression and osteochondral differentiation in PRECs. TGF-β1 alone could effectively promote EMT, but it had no effect on osteochondral differentiation in NRK cells (Rat kidney epithelial cell line). Stimulation with Ca²⁺ alone did not accelerate differentiation of NRK. Co-incubation of extracellular Ca²⁺ and TGF-β1 synergistically promotes EMT and osteochondral differentiation in NRK control cells. Our data supplied a novel view that the pathogenesis of calcium stone development may be associated with synergic effects of TGF-β1 and Ca²⁺, which promote EMT and osteochondral differentiation via Wnt11 and the L-type calcium channel.     

Dityrosine Crosslinking of Collagen and Amyloid-β Peptides Is Formed by Vitamin B12 Deficiency-Generated Oxidative Stress in Caenorhabditis elegans

(1) Background: Vitamin B₁₂ deficiency in Caenorhabditis elegans results in severe oxidative stress and induces morphological abnormality in mutants due to disordered cuticle collagen biosynthesis. We clarified the underlying mechanism leading to such mutant worms due to vitamin B₁₂ deficiency. (2) Results: The deficient worms exhibited decreased collagen levels of up to approximately 59% compared with the control. Although vitamin B₁₂ deficiency did not affect the mRNA expression of prolyl 4-hydroxylase, which catalyzes the formation of 4-hydroxyproline involved in intercellular collagen biosynthesis, the level of ascorbic acid, a prolyl 4-hydroxylase coenzyme, was markedly decreased. Dityrosine crosslinking is involved in the extracellular maturation of worm collagen. The dityrosine level of collagen significantly increased in the deficient worms compared with the control. However, vitamin B₁₂ deficiency hardly affected the mRNA expression levels of bli-3 and mlt-7, which are encoding crosslinking-related enzymes, suggesting that deficiency-induced oxidative stress leads to dityrosine crosslinking. Moreover, using GMC101 mutant worms that express the full-length human amyloid β, we found that vitamin B₁₂ deficiency did not affect the gene and protein expressions of amyloid β but increased the formation of dityrosine crosslinking in the amyloid β protein. (3) Conclusions: Vitamin B₁₂-deficient wild-type worms showed motility dysfunction due to decreased collagen levels and the formation of highly tyrosine-crosslinked collagen, potentially reducing their flexibility. In GMC101 mutant worms, vitamin B₁₂ deficiency-induced oxidative stress triggers dityrosine-crosslinked amyloid β formation, which might promote its stabilization and toxic oligomerization.