Regulation Of Responses Research Articles

Phosphoproteomic analysis uncovers phosphorylated proteins in response to salicylic acid and N-hydroxypipecolic acid in Arabidopsis.

The phytohormone salicylic acid (SA) serves as a crucial signaling molecule within the realm of plant immunity, playing an indispensable role in both local and systemic acquired resistance (SAR). N-hydroxypipecolic acid (NHP), a derivative of L-lysine, is integral to the induction of SAR. Recent investigations have illuminated the intricate manner in which NHP orchestrates the establishment of SAR in conjunction with the immune signal SA. To further explore the mechanisms governing the synergistic regulation of SAR by SA and NHP, we conducted an extensive phosphoproteomic analysis aimed at identifying the phosphoproteins modulated either commonly or uniquely by SA and NHP, employing a phosphoproteomics platform built upon high-resolution mass spectrometry. Our study revealed a total of 133 phosphopeptides, derived from 115 distinct proteins, exhibiting exclusive responsiveness to NHP treatment. In contrast, 229 phosphopeptides sourced from 204 proteins demonstrated exclusive sensitivity to SA treatment. Additionally, the phosphorylation status of 215 proteins, including numerous kinases, phosphatases, transcription factors, and proteins implicated in membrane trafficking, was commonly modulated by both SA and NHP. This investigation offers detailed insights into the key phosphoproteins influenced either collectively or specifically by SA and NHP, thereby enabling further exploration of the mechanisms underlying the synergistic regulation of immune responses orchestrated by these two potent molecules.

PRMT5-regulated splicing of DNA repair genes drives chemoresistance in breast cancer stem cells.

Breast cancer stem cells (BCSCs) are a rare cell population that is responsible for tumour initiation, metastasis and chemoresistance. Despite this, the mechanism by which BCSCs withstand genotoxic stress is largely unknown. Here, we uncover a pivotal role for the arginine methyltransferase PRMT5 in mediating BCSC chemoresistance by modulating DNA repair efficiency. Mechanistically, we identify PRMT5 as a major regulator of DNA damage response (DDR) gene splicing in BCSCs, particularly those integral to the Fanconi Anaemia and homologous recombination pathways, with PRMT5 inhibition synergising with chemotherapy to promote BCSC apoptosis. A comparison of BCSCs and their bulk cell progeny identified some shared (ATM, DDX11, EXO1, FAN1, SLX4) but many unique (ATR, RAD17, RAD51D, RUVBL1) PRMT5-dependent alternative DDR splicing events. Surprisingly, these skipped exons and retained intron events rarely lead to substantial gene expression repression, suggesting that PRMT5 inhibition predominantly results in nuclear detention of intron-containing transcripts and the production of non-canonical isoforms with compromised protein function. Since many genes within the same DDR pathway undergo deregulated splicing, this study thus reveals additional points of vulnerability and alternative combination drug strategies that could improve the therapeutic efficacy of PRMT5 inhibitors to promote BCSC eradication.

EMSA-SELEX-seq method for analysis of binding site sequences in DNA-protein complexes

The BOB1 protein (OBF1, OCA-B) is a transcriptional coactivator of two POU domain proteins — OCT1, expressed in all cells, and lymphoid-specific OCT2. The interaction of BOB1 with OCT1/2 plays an important role in the regulation of immune responses in both physiological and pathological contexts. BOB1 is known to form a ternary complex with OCT1/2 bound to DNA in monomeric and certain dimeric configurations, changing the sequence specificity of the binding. To analyze DNA sequences from these complexes, in this work we proposed the EMSA-SELEX-seq method, based on the separation of OCT/BOB1 complexes of various compositions in a non-denaturing polyacrylamide gel (EMSA) followed by the isolation and amplification of the oligonucleotides that they contain (SELEX). Based on several rounds of the enrichment followed by the NGS sequencing and bioinformatics analysis, the DNA sequences were determined and the relevance of this approach was confirmed. Thus, the proposed EMSA-SELEX-seq method allows the analysis of DNA sequences in DNA-protein complexes with varying dimensions of its protein components.

ENPP1/CD203a-targeting heavy-chain antibody reveals cell-specific expression on human immune cells

ENPP1/CD203a is a membrane-bound ectonucleotidase capable of hydrolyzing ATP, cGAMP and other substrates. Its enzymatic activity plays an important role in the balance of extracellular adenine nucleotides and the modulation of purinergic signaling, in soft tissue calcification, and in the regulation of the cGAS/STING pathway. However, a detailed analysis of ENPP1 surface expression on human immune cells has not been performed. Here, we selected VHH domains from human ENPP1-immunized alpacas to generate heavy-chain antibodies targeting ENPP1, and analyzed cell surface expression on all circulating immune cell subsets using flow cytometry. We find high expression of ENPP1 in CD141high conventional dendritic cells (cDC1), while ENPP1 was not detectable on other dendritic cells and monocytes. In the lymphocytic compartment, only CD56bright natural killer cells and mucosal-associated invariant T cells (MAIT) express ENPP1. In contrast, all other T cell subpopulations, CD56dim natural killer cells and B lymphocytes do not or only minimally express ENPP1. In summary, we describe highly cell type-specific expression of ENPP1 in the immune system using a newly generated heavy-chain antibody. This reagent will help to decipher the function of ENPP1 in the regulation of the immune response, allow a quick identification of ENPP1-deficiency and of ENPP1-positive tumors, and constitutes the basis for targeted anti-tumor intervention.

Pseudomonas oryzihabitans D1-104/3 and P. gessardii C31-106/3 differentially modulate the antioxidative response of duckweed (Lemna minor L.) to salt stress

IntroductionThe common duckweed (L. minor) is a model organism for investigation of plant physiology, especially stress-related responses. Its two physiological characteristics are of special interest: (1) salt-stressed duckweeds may accumulate starch, a precursor for biofuel; (2) duckweeds are associated with various beneficial (plant-growth promoting, PGP) bacterial strains. In this paper, we analyzed the role of two bacterial strains: Pseudomonas oryzihabitans D1-104/3 and P. gessardii C31-106/3 in regulation of duckweed's growth and antioxidative responses to salt (10 and 100 mM NaCl) and hypothesized that they alleviate salt-induced oxidative stress.MethodsFresh and dry weight, frond number, photosynthetic pigments, malondialdehyde (MDA) and hydrogen peroxide (H2O2), ascorbic acid (AsA), proline, total polyphenol (TPC) and starch content, as well as antioxidant capacity and antioxidant enzymatic activity were measured after 14 days. Fluorescence microscopy was used to visualize bacterial presence on duckweeds.ResultsFluorescence microscope revealed that Pseudomonas bacteria colonized all duckweed surfaces. The doubling time of duckweeds inoculated with P. gessardii C31-106/3 was significantly longer. Additionally, at 0 and 10 mM NaCl, this strain decreased chlorosis in duckweeds. Moreover, P. gessardii C31-106/3 increased dry-to-fresh-weight ratio, proline, chlorophyll a, b and carotenoid content at 100 mM, as well as AsA content in plants in NaCl-free medium, while P. oryzihabitans D1-104/3 increased AsA at 100 mM NaCl. Both bacterial strains decreased lipid peroxidation, while P. gessardii C31-106/3 increased and P. oryzihabitans D1-104/3 decreased H2O2 content at 100 mM and 0 mM NaCl, respectively. Bacteria significantly increased TPC and antioxidant capacity at 100 mM NaCl, particularly P. oryzihabitans D1-104/3. After 14 days, the SOD and POX activities were at the same level in all samples. At 100 mM NaCl, CAT activity was increased in all plants.DiscussionThe results of this study show that two Pseudomonas strains had markedly different effects on duckweed: while P. oryzihabitans D1-104/3 supported growth, P. gessardii C31-106/3 prioritized salt stress tolerance in duckweeds.

Nucleotide-binding oligomerization domain 1 (NOD1) regulates microglial activation in pseudorabies virus infection

The primary cause of viral encephalitis (VE) is invasion of the central nervous system (CNS) by the virus, which leads to neuroinflammation and poses a significant threat to global public health. Microglia, as CNS-resident macrophages, play a crucial role in neuroinflammation and are often identified as the preferred target for the prevention or treatment of VE. In this study, we used pseudorabies virus (PRV)-induced VE in mice and pigs as a model to investigate the regulation of microglial responses during viral encephalitis and explored the mechanism of microglial activation. Cellular experiments revealed that microglial activation was accompanied by cell migration, characteristic morphological changes, phagocytosis, inflammatory cytokine production, and antigen presentation. Transcriptome analysis revealed that genes related to inflammation in PRV-infected BV2 cells were significantly enriched. The expression of the NOD1 gene in BV2 cells was significantly increased during PRV infection, after which NOD1 in BV2 cells was silenced by siRNA and overexpressed via a plasmid. NOD1 was found to be involved in the secretion of cytokines in BV2 cells by regulating the MAPK/NF-κB signalling pathway. Mouse and pig experiments have shown that NOD1 is involved in the secretion of cytokines by microglia by regulating the MAPK/NF-κB signalling pathway during PRV infection.

重構公共與私人界限：公共健康作為人口層面視角的形而上學框架——對麥凱“公共健康倫理學：ー種藝術的現狀”ー文的評論

LANGUAGE NOTE | Document text in Chinese; abstract also in English. This commentary explores Kathryn MacKay’s concept of public health as a metaphysical framework that shifts private issues, such as smoking and domestic violence, into the area of public health. Using a third-person, population-level perspective, this paper examines the ethical basis for reframing such behaviours as public health issues, advocating for stricter regulation of smoking and systemic responses to domestic violence. MacKay’s framework offers valuable support for advancing comprehensive public health policies, which is particularly relevant in China.

Capsicum annum NAC transcription factor CaNAC12 functions as a positive regulator of the stress response to salt, drought, and Botrytis cinerea in Nicotiana benthamiana

Capsicum annum NAC transcription factor CaNAC12 functions as a positive regulator of the stress response to salt, drought, and Botrytis cinerea in Nicotiana benthamiana

Transcription factors CpSPL5 and CpSPL8 negatively regulate salt tolerance in Codonopsis pilosula by inhibiting SOS pathway.

Environmental stresses such as salt and drought severely affect plant growth and development. SQUAMOSA-promoter binding protein-like (SPL) transcription factors (TFs) play critical roles in the regulation of diverse processes; however, reports describing the SPL regulation of plant responses to abiotic stress are relatively few. In this study, two stress-responsive TFs from Codonopsis pilosula (CpSPL5 and CpSPL8) are reported, which confer salt stress sensitivity. CpSPL5 and CpSPL8 are expressed in almost all tissues and localized in the nucleus, where the CpSPL5 transcript level is relatively higher than that of CpSPL8. Their expression levels are significantly suppressed in hairy roots treated with ABA, NaCl, PEG-6000, and under high temperature stress. Compared with the control, CpSPL5, or CpSPL8-overexpressed hairy roots increased salt stress sensitivity, and exhibited higher levels of O2- and MDA, as well as lower superoxide dismutase and peroxidase activities. Further, the CpSPL5 or CpSPL8 interference transgenic hairy roots enhanced salt tolerance and exhibited contrasting phenotype and antioxidant indices. Although all genotypes revealed significantly increased Na+ and decreased K+ contents under salt stress, the physiological indicators of CpSPL5 or CpSPL8-interference transgenic hairy roots could be partially restored, where CpSPL5 was more sensitive to salt stress than CpSPL8. A yeast one-hybrid and dual-luciferase assay revealed that CpSPL5 and CpSPL8 directly targeted and inhibited the expression of CpSOS2 in the salt overly sensitive (SOS) pathway, which promoted salt stress sensitivity. Our findings suggest that CpSPL5 and CpSPL8 served as negative regulators of salt tolerance, which indicate that members of the SPL family participate in the plant SOS pathway.

SEMA3G-NRP1 Signaling Functions as an Immune Checkpoint that Enables Tumor Immune Evasion by Impairing T Cell Cytotoxicity.

T cells within the tumor microenvironment frequently exhibit dysfunctional characteristics that compromise their ability to elicit both innate and therapeutic-induced immune responses. Regulators of immune dysfunction represent therapeutic targets to activate anti-tumor immunity. In this study, we identified semaphorin 3G (SEMA3G) as a key regulator of immune responses in cancer. SEMA3G was widely upregulated in diverse human cancers, and its expression was positively correlated with tumor progression. SEMA3G acted as a ligand that inhibited the activation and functionality of T cells. A comprehensive receptor screening approach demonstrated that SEMA3G exhibited a significantly stronger affinity for neuropilin NRP1 compared to NRP2. Furthermore, SEMA3G primarily impeded T-cell functions via NRP1. Disruption of SEMA3G using CRISPR/Cas9 technology or blockade with a neutralizing antibody effectively restored the cytotoxicity of CD8+ T cells and inhibited the growth of tumors in vivo. This research underscores the role of SEMA3G in T-cell dysfunction within tumors and proposes a targeting SEMA3G as a cancer immunotherapeutic strategy.

TRPM7 activity drives human CD4 T-cell activation and differentiation in a magnesium dependent manner.

T lymphocyte activation is a crucial process in the regulation of innate and adaptive immune responses. The ion channel-kinase TRPM7 has previously been implicated in cellular Mg2+ homeostasis, proliferation, and immune cell modulation. Here, we show that pharmacological and genetic silencing of TRPM7 leads to diminished human CD4 T-cell activation and proliferation following TCR mediated stimulation. In both primary human CD4 T cells and CRISPR/Cas-9 engineered Jurkat T cells, loss of TRPM7 led to altered Mg2+ homeostasis, Ca2+ signaling, reduced NFAT translocation, decreased IL-2 secretion and ultimately diminished proliferation and differentiation. While the activation of primary human CD4 T cells was dependent on TRPM7, polarization of naïve CD4 T cells into regulatory T cells (Treg) was not. Taken together, these results highlight TRPM7 as a key protein of cellular Mg2+ homeostasis and CD4 T-cell activation. Its role in lymphocyte activation suggests therapeutic potential for TRPM7 in numerous T-cell mediated diseases.

A conserved nuclear factor YC subunit, NF-YC3, is essential for arbuscule development.

Establishing reciprocal symbiosis with arbuscular mycorrhizal (AM) fungi is an important evolutionary strategy of most terrestrial plants to adapt to environmental stresses, especially phosphate (Pi) deficiencies. Identifying the key genes essential for AM symbiosis in plants and dissecting their functional mechanisms will be helpful for the breeding of new crop varieties with enhanced nutrient uptake efficiency. Here, we report a nuclear factor YC subunit-encoding gene, OsNF-YC3, whose expression is specifically induced in arbuscule-containing cells, plays an essential role in AM symbiosis. Knockout of OsNF-YC3 resulted in stunted arbuscule morphology and substantially decreased P accumulation, while overexpressing OsNF-YC3 enhanced mycorrhization and Pi uptake efficiency. OsNF-YC3 is directly regulated by OsPHRs, the major regulators of Pi starvation responses. Chromatin immunoprecipitation sequencing analysis uncovered multiple genes with crucial roles in arbuscule development as its potential downstream targets, including the AM-specific Pi transporter gene OsPT11. OsNF-YC3 can form a heterotrimer with the other two NF-Y subunits, OsNF-YA11 and OsNF-YB11, in yeast. Loss of OsNF-YA11 function also severely impaired arbuscule development in its mutants. Overall, our results highlight an essential role of OsNF-YC3 and its potential interacting NF-Y subunit, OsNF-YA11, in regulating AM symbiosis and arbuscule development.

Cytokine Modulation with Biomaterials: Engineering the Immune Response for Tissue Repair and Regeneration

Cytokines are key regulators of the immune response and play critical roles in tissue repair and regeneration. Modulating the cytokine environment at sites of injury can improve healing by reducing chronic inflammation and promoting tissue remodeling. Biomaterials have emerged as effective platforms for delivering cytokines in a controlled and localized manner, providing spatial and temporal regulation of the immune response. This review discusses various biomaterial-based strategies for cytokine modulation, including hydrogels, nanoparticles, and scaffolds, each designed to influence immune cell behavior and enhance tissue regeneration. Biomaterials can be engineered to deliver specific cytokines, such as interleukin-10 (IL-10) or transforming growth factor-beta (TGF-β), which help suppress inflammation and promote the differentiation of stem cells or other progenitor cells. Applications of cytokine-modulating biomaterials in wound healing, bone regeneration, cardiac repair, and nerve regeneration are explored. Additionally, challenges such as achieving precise cytokine release, maintaining cytokine stability, and the complexity of immune regulation are addressed. Advances in biomaterial design hold great potential for developing “smart” systems capable of adjusting cytokine delivery based on the evolving tissue environment. The use of cytokine-modulating biomaterials represents a promising approach to improving clinical outcomes in regenerative medicine and tissue engineering. Keywords: Cytokine modulation, Biomaterial, Tissue regeneration, Immune response, Inflammation, Controlled release

Menisoxoisoaporphine A, a novel oxoisoaporphine alkaloid from Menispermi Rhizoma, inhibits inflammation by targeting PDE4B.

Dysregulated and excessive inflammatory reactions can lead to tissue damage, which is the underlying cause of most human diseases. Menisoxoisoaporphine A (MA), a novel oxoisoaporphine alkaloid, was obtained from Menispermi Rhizoma, a traditional Chinese medicinal herb used in the treatment of inflammatory conditions in clinical practice. This suggests that MA has very promising potential for the development of anti-inflammatory therapeutics. Hence, this study aims to investigate the anti-inflammatory effects and underlying mechanisms of MA. The anti-inflammatory effects of MA were evaluated in lipopolysaccharide (LPS)-induced mouse macrophage RAW264.7 cells. Its underlying mechanisms were explored through RNA sequencing and Western blotting. The binding modes and interactions sites between MA and phosphodiesterase 4B (PDE4B) were predicted using molecular docking and validated by molecular dynamics simulation. MA treatment significantly reduced LPS-induced morphological changes, inflammatory cytokine relesae, and proinflammatory genes expression in RAW264.7 cells compared to the LPS-induced controls. Transcriptome sequencing analysis suggested that PDE4B might be a key target for MA to exert its therapeutic effect. Mechanismly, MA directly acted on Tyr405 site of PDE4B, thus leading to a sustained elevation of the cyclic adenosine monophosphate (cAMP) levels, which subsequently inactivated NF-κB signaling pathway by phosphorylating protein kinase A (PKA). MA inhibited the NF-κB-mediated inflammatory response depending on PDE4B. MA, a natural and novel compound, exerted anti-inflammatory effects in LPS-induced RAW264.7 macrophage cells. It demonstrated a strong binding ability to the Tyr405 sites of PDE4B, thereby inhibiting NF-κB signaling pathway by regulating the cAMP-PKA axis. Elucidating the interaction between MA and PDE4B holds significant potential for the advancement of innovative therapeutic strategies aimed at inflammatory diseases. By strategically modulating this interaction, it may be feasible to achieve more precise regulation of inflammatory responses, thereby offering promising therapeutic benefits for conditions such as rheumatoid arthritis, asthma, and inflammatory bowel disease.

Genome-wide identification of HIPP and mechanism of SlHIPP4/7/9/21/26/32 mediated phytohormones response to Cd, osmotic, and salt stresses in tomato

Heavy-metal-associated isoprenylated plant proteins (HIPPs) contributed to abiotic tolerance in vascular plants. Up to now, the HIPP gene family of tomato (Solanum lycopersicum L.) had not been thoroughly understood. In the present study, 34 SlHIPP genes were identified from the tomato genome using the Hidden Markov Model (HMM). The phylogenetic analysis revealed that the evolution of SlHIPPs was highly conserved. The cis-acting element analysis indicated that SlHIPP genes might be involved in phytohormones and abiotic stresses. We constructed venn diagram with 17 genes containing stress-related motifs as well as 15 genes and 19 genes expressing in leaves and roots in RNA-seq data, suggesting that SlHIPP4/7/9/21/26/32 were selected as candidate genes for study. The quantitative real-time PCR (qRT-PCR) analysis showed that 6 candidate genes were indicated to be involved in osmotic and salt stress tolerance and SlHIPP7/21/26/32 responded to cadmium (Cd) tolerance. The virus-induced silencing of 6 candidate genes caused growth inhibition in stress conditions, further illustrating that 6 candidate genes played a positive role in abiotic conditions. Importantly, the phytohormone analysis implied that 6 candidate genes mediated abscisic acid (ABA), salicylic acid (SA), gibberellin (GA3), auxin (IAA), or methyl jasmonate (MeJA) responses to Cd, osmotic, or salt stress tolerance. These findings indicated that SlHIPP4/7/9/21/26/32 were key regulators of abiotic stress responses in tomato seedlings, functioning through multiple phytohormone pathways.

318 Non-Segmental Vitiligo in Older Patients is Associated with Increased Activation and Decreased Regulation of the Systemic Immune Response, Independent of Disease Duration

318 Non-Segmental Vitiligo in Older Patients is Associated with Increased Activation and Decreased Regulation of the Systemic Immune Response, Independent of Disease Duration

Characterization of radiations‐induced genomic structural variations in Arabidopsis thaliana

SUMMARYDNA, is assaulted by endogenous and exogenous agents that lead to the formation of damage. In order to maintain genome integrity DNA repair pathways must be efficiently activated to prevent mutations and deleterious chromosomal rearrangements. Conversely, genome rearrangement is also necessary to allow genetic diversity and evolution. The antagonist interaction between maintenance of genome integrity and rearrangements determines genome shape and organization. Therefore, it is of great interest to understand how the whole linear genome structure behaves upon formation and repair of DNA damage. For this, we used long reads sequencing technology to identify and to characterize genomic structural variations (SV) of wild‐type Arabidopsis thaliana somatic cells exposed either to UV‐B, to UV‐C or to protons irradiations. We found that genomic regions located in heterochromatin are more prone to form SVs than those located in euchromatin, highlighting that genome stability differs along the chromosome. This holds true in Arabidopsis plants deficient for the expression of master regulators of the DNA damage response (DDR), ATM (Ataxia‐telangiectasia‐mutated) and ATR (Ataxia‐telangiectasia‐mutated and Rad3‐related), suggesting that independent and alternative surveillance processes exist to maintain integrity in genic regions. Finally, the analysis of the radiations‐induced deleted regions allowed determining that exposure to UV‐B, UV‐C and protons induced the microhomology‐mediated end joining mechanism (MMEJ) and that both ATM and ATR repress this repair pathway.

Pleiotrophin modulates acute and long-term LPS-induced neuroinflammatory responses and hippocampal neurogenesis

The hippocampus is one of the most vulnerable regions affected in disorders characterized by overt neuroinflammation such as neurodegenerative diseases. Pleiotrophin (PTN) is a neurotrophic factor that modulates acute neuroinflammation in different contexts. PTN is found highly upregulated in the brain in different chronic disorders characterized by neuroinflammation, suggesting an important role in the modulation of sustained neuroinflammation. To test this hypothesis, we studied the acute and long-term effects of a single lipopolysaccharide (LPS; 5mg/kg) administration in Ptn+/+ and Ptn-/- mice, and in mice with Ptn-overexpression (Ptn-Tg). Endogenous PTN levels proportionally modulate LPS-induced increase in TNF-α plasma levels one hour after treatment. In the dentate gyrus (DG) of the hippocampus, a lower percentage of DCX+ cells were detected in saline-treated Ptn-/- mice compared to Ptn+/+ mice, suggesting a crucial role of PTN in the maintenance of hippocampal neuronal progenitors. The data show that PTN overexpression tends to potentiate acute microglial responses in the DG 16hours after LPS treatment. Remarkably, a significant increase in the number of neuronal progenitors together with astrogliosis was detected 10 months after a single injection of LPS treatment in wild type mice. However, these LPS-induced long-term effects were prevented in Ptn-/- and Ptn-Tg mice, suggesting that PTN modulates LPS-induced long-term neurogenesis changes and astrocytic response in the hippocampus. The data presented here suggest that endogenous PTN levels are crucial in the regulation of acute LPS-induced systemic and hippocampal microglial responses in young mice. Furthermore, our findings provide evidence of the key role of PTN in the regulation of long-term LPS effects on astrocytic response and neurogenesis in the hippocampus.

320 The implication of RNA modification m6Am in the regulation of cellular response in human primary keratinocytes suggests its potential importance for the development of psoriasis

320 The implication of RNA modification m6Am in the regulation of cellular response in human primary keratinocytes suggests its potential importance for the development of psoriasis

3D confinement alters smooth muscle cell responses to chemical and mechanical cues.

Smooth muscle cell (SMC) phenotypic switching is a hallmark of many vascular diseases. Although prior work has established that chemical and mechanical cues contribute to SMC phenotypic switching, the impact of three-dimensional (3D) confinement on this process remains elusive. Yet, in vivo, arterial SMCs reside within confined environments. In this study, we designed a microfluidic assay to investigate the interplay between 3D confinement and different environmental stimuli in SMC function. Our results show that tightly, but not moderately, confined SMCs acquire a contractile phenotype when exposed to collagen I. Elevated compressive forces induced by hydrostatic pressure abolish this upregulation of the contractile phenotype and compromise SMC survival, particularly in tightly confined spaces. Transforming growth factor beta 1, which promotes the contractile state in moderate confinement, fails to enhance the contractility of tightly confined cells. Fibronectin and engagement of cadherin 2 suppress the contractile phenotype of SMCs regardless of the degree of confinement. In contrast, homophilic engagement of cadherin 11 upregulates SMC-specific genes and enhances contractility in both moderately and tightly confined cells. Overall, our work introduces 3D confinement as a regulator of SMC phenotypic responses to chemical and mechanical signals.