Non-canonical Pathway Research Articles

Large-scale phosphoproteomics reveals activation of the MAPK/GADD45β/P38 axis and cell cycle inhibition in response to BMP9 and BMP10 stimulation in endothelial cells

BackgroundBMP9 and BMP10 are two major regulators of vascular homeostasis. These two ligands bind with high affinity to the endothelial type I kinase receptor ALK1, together with a type II receptor, leading to the direct phosphorylation of the SMAD transcription factors. Apart from this canonical pathway, little is known. Interestingly, mutations in this signaling pathway have been identified in two rare cardiovascular diseases, hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension.MethodsTo get an overview of the signaling pathways modulated by BMP9 and BMP10 stimulation in endothelial cells, we employed an unbiased phosphoproteomic-based strategy. Identified phosphosites were validated by western blot analysis and regulated targets by RT-qPCR. Cell cycle analysis was analyzed by flow cytometry.ResultsLarge-scale phosphoproteomics revealed that BMP9 and BMP10 treatment induced a very similar phosphoproteomic profile. These BMPs activated a non-canonical transcriptional SMAD-dependent MAPK pathway (MEKK4/P38). We were able to validate this signaling pathway and demonstrated that this activation required the expression of the protein GADD45β. In turn, activated P38 phosphorylated the heat shock protein HSP27 and the endocytosis protein Eps15 (EGF receptor pathway substrate), and regulated the expression of specific genes (E-selectin, hyaluronan synthase 2 and cyclooxygenase 2). This study also highlighted the modulation in phosphorylation of proteins involved in transcriptional regulation (phosphorylation of the endothelial transcription factor ERG) and cell cycle inhibition (CDK4/6 pathway). Accordingly, we found that BMP10 induced a G1 cell cycle arrest and inhibited the mRNA expression of E2F2, cyclinD1 and cyclinA1.ConclusionsOverall, our phosphoproteomic screen identified numerous proteins whose phosphorylation state is impacted by BMP9 and BMP10 treatment, paving the way for a better understanding of the molecular mechanisms regulated by BMP signaling in vascular diseases.

Metabolic rewiring enables ammonium assimilation via a non-canonical fumarate-based pathway.

Glutamate serves as the major cellular amino group donor. In Bacillus subtilis, glutamate is synthesized by the combined action of the glutamine synthetase and the glutamate synthase (GOGAT). The glutamate dehydrogenases are devoted to glutamate degradation invivo. To keep the cellular glutamate concentration high, the genes and the encoded enzymes involved in glutamate biosynthesis and degradation need to be tightly regulated depending on the available carbon and nitrogen sources. Serendipitously, we found that the inactivation of the ansR and citG genes encoding the repressor of the ansAB genes and the fumarase, respectively, enables the GOGAT-deficient B. subtilis mutant to synthesize glutamate via a non-canonical fumarate-based ammonium assimilation pathway. We also show that the de-repression of the ansAB genes is sufficient to restore aspartate prototrophy of an aspB aspartate transaminase mutant. Moreover, in the presence of arginine, B. subtilis mutants lacking fumarase activity show a growth defect that can be relieved by aspB overexpression, by reducing arginine uptake and by decreasing the metabolic flux through the TCA cycle.

The immunopathological landscape of human pre-TCRα deficiency: From rare to common variants.

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre-α T cell receptor (pre-TCRα) expression. Low circulating naive αβ T cell counts at birth persisted over time, with normal memory αβ and high γδ T cell counts. Their TCRα repertoire was biased, which suggests that noncanonical thymic differentiation pathways can rescue αβ T cell development. Only a minority of these individuals were sick, with infection, lymphoproliferation, and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naive αβ T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αβ T cells, autoimmune conditions were more frequent in these patients compared with the general population.

The Mechanism of Pyroptosis and Its Application Prospect in Diabetic Wound Healing.

Pyroptosis defines a form of pro-inflammatory-dependent programmed cell death triggered by gasdermin proteins, which creates cytoplasmic pores and promotes the activation and accumulation of immune cells by releasing several pro-inflammatory mediators and immunogenic substances upon cell rupture. Pyroptosis comprises canonical (mediated by Caspase-1) and non-canonical (mediated by Caspase-4/5/11) molecular signaling pathways. Numerous studies have explored the contributory roles of inflammasome and pyroptosis in the progression of multiple pathological conditions such as tumors, nerve injury, inflammatory diseases and metabolic disorders. Accumulating evidence indicates that the activation of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome results in the activation of pyroptosis and inflammation. Current evidence suggests that pyroptosis-dependent cell death plays a progressive role in the development of diabetic complications including diabetic wound healing (DWH) and diabetic foot ulcers (DFUs). This review presents a brief overview of the molecular mechanisms underlying pyroptosis and addresses the current research on pyroptosis-dependent signaling pathways in the context of DWH. In this review, we also present some prospective therapeutic compounds/agents that can target pyroptotic signaling pathways, which may serve as new strategies for the effective treatment and management of diabetic wounds.

Age-associated accumulation of B cells promotes macrophage inflammation and inhibits lipolysis in adipose tissue during sepsis

Non-canonical lipolysis induced by inflammatory cytokines or Toll-like receptor ligands is required for the regulation of inflammation during endotoxemia and sepsis. Canonical lipolysis induced by catecholamines declines during aging due to factors including an expansion of lymphocytes, pro-inflammatory macrophage polarization, and an increase in chronic low-grade inflammation; however, the extent to which the non-canonical pathway of lipolysis is active and impacted by immune cells during aging remains unclear. Therefore, we aimed to define the extent to which immune cells from old mice influence non-canonical lipolysis during sepsis. We identified age-associated impairments of non-canonical lipolysis and an accumulation of dysfunctional B1 B cells in the visceral white adipose tissue (vWAT) of old mice. Lifelong deficiency of B cells results in restored non-canonical lipolysis and reductions in pro-inflammatory macrophage populations. Our study suggests that targeting the B cell-macrophage signaling axis may resolve metabolic dysfunction in aged vWAT and attenuate septic severity in older individuals.

Simvastatin Abrogates TGF-β Induced Stimulation of Normal Human Dermal Fibroblast Collagen Gene Expression in Culture

A potent stimulation of fibroblast collagen production is one of the crucial pleotropic effects of transforming growth factor β (TGF-β) and has been considered to play a crucial role in the pathogenesis of fibrotic diseases including Systemic Sclerosis and Pulmonary Fibrosis. This complex process involves numerous intracellular reactions mediated by canonical Smad-dependent or non-canonical pathways that transduce the extracellular stimuli into the nucleus. Here, we demonstrated that Simvastatin, a widely used statin, induces a potent inhibition of TGF-β1 profibrotic effects in cultured normal human dermal fibroblasts, and studied the molecular mechanisms involved in these effects. We also examined Simvastatin modulation of TGF-β1 induced fibroblast to myofibroblast transition. Normal human dermal fibroblasts were cultured with various concentrations of Simvastatin in the presence or absence of TGF-β1 (10ng/ml) for 24, 48, and 72 h. The effects of Simvastatin on TGF-β1 stimulation of COL1A1 expression and type 1 collagen production were examined. Assessment of Smad2/3 and Erk1/2 phosphorylation, chromatin immunoprecipitation assays for Sp1 transcription factor binding to the COL1A1 proximal promoter, siRNA-mediated RhoA knockdown, and F-actin immunofluorescence microscopy was performed to examine the molecular mechanisms involved.

Unravelling the Road to Recovery: Mechanisms of Wnt Signalling in Spinal Cord Injury.

Spinal cord injury (SCI) is a complex neurodegenerative pathology that consistently harbours a poor prognostic outcome. At present, there are few therapeutic strategies that can halt neuronal cell death and facilitate functional motor recovery. However, recent studies have highlighted the Wnt pathway as a key promoter of axon regeneration following central nervous system (CNS) injuries. Emerging evidence also suggests that the temporal dysregulation of Wnt may drive cell death post-SCI. A major challenge in SCI treatment resides in developing therapeutics that can effectively target inflammation and facilitate glial scar repair. Before Wnt signalling is exploited for SCI therapy, further research is needed to clarify the implications of Wnt on neuroinflammation during chronic stages of injury. In this review, an attempt is made to dissect the impact of canonical and non-canonical Wnt pathways in relation to individual aspects of glial and fibrotic scar formation. Furthermore, it is also highlighted how modulating Wnt activity at chronic time points may aid in limiting lesion expansion and promoting axonal repair.

Primary cilia promote the differentiation of human neurons through the WNT signaling pathway.

Primary cilia emanate from most human cell types, including neurons. Cilia are important for communicating with the cell's immediate environment: signal reception and transduction to/from the ciliated cell. Deregulation of ciliary signaling can lead to ciliopathies and certain neurodevelopmental disorders. In the developing brain cilia play well-documented roles for the expansion of the neural progenitor cell pool, while information about the roles of cilia during post-mitotic neuron differentiation and maturation is scarce. We employed ciliated Lund Human Mesencephalic (LUHMES) cells in time course experiments to assess the impact of ciliary signaling on neuron differentiation. By comparing ciliated and non-ciliated neuronal precursor cells and neurons in wild type and in RFX2 -/- mutant neurons with altered cilia, we discovered an early-differentiation "ciliary time window" during which transient cilia promote axon outgrowth, branching and arborization. Experiments in neurons with IFT88 and IFT172 ciliary gene knockdowns, leading to shorter cilia, confirm these results. Cilia promote neuron differentiation by tipping WNT signaling toward the non-canonical pathway, in turn activating WNT pathway output genes implicated in cyto-architectural changes. We provide a mechanistic entry point into when and how ciliary signaling coordinates, promotes and translates into anatomical changes. We hypothesize that ciliary alterations causing neuron differentiation defects may result in "mild" impairments of brain development, possibly underpinning certain aspects of neurodevelopmental disorders.

Bhlhe40 deficiency attenuates LPS-induced acute lung injury through preventing macrophage pyroptosis

BackgroundAcute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) as common life-threatening lung diseases with high mortality rates are mostly associated with acute and severe inflammation in lungs. Recently, increasing evidence supports activated inflammation and gasdermin D (GSDMD)-mediated pyroptosis in macrophage are closely associated with ALI. Basic helix-loop-helix family member e40 (Bhlhe40) is a transcription factor that is comprehensively involved in inflammation. However, there is little experimental evidence connecting Bhlhe40 and GSDMD-driven pyroptosis. The study sought to verify the hypothesis that Bhlhe40 is required for GSDMD-mediated pyroptosis in lipopolysaccharide (LPS)-induced inflammatory injury.MethodWe performed studies using Bhlhe40-knockout (Bhlhe40 −/−) mice, small interfering RNA (siRNA) targeting Bhlhe40 and pyroptosis inhibitor disulfiram to investigate the potential roles of Bhlhe40 on LPS-induced ALI and the underlying mechanisms.ResultsBhlhe40 was highly expressed in total lung tissues and macrophages of LPS-induced mice. Bhlhe40−/− mice showed alleviative lung pathological injury and inflammatory response upon LPS stimulation. Meanwhile, we found that Bhlhe40 deficiency significantly suppressed GSDMD-mediated pyroptosis in macrophage in vivo and in vitro. By further mechanistic analysis, we demonstrated that Bhlhe40 deficiency inhibited GSDMD-mediated pyroptosis and subsequent ALI by repressing canonical (caspase-1-mediated) and non-canonical (caspase-11-mediated) signaling pathways in vivo and in vitro.ConclusionThese results indicate Bhlhe40 is required for LPS-induced ALI. Bhlhe40 deficiency can inhibit GSDMD-mediated pyroptosis and therefore alleviate ALI. Targeting Bhlhe40 may be a potential therapeutic strategy for LPS-induced ALI.

Targeting EphA2: a promising strategy to overcome chemoresistance and drug resistance in cancer.

Erythropoietin-producing hepatocellular A2 (EphA2) is a vital member of the Eph tyrosine kinase receptor family and has been associated with developmental processes. However, it is often overexpressed in tumors and correlates with cancer progression and worse prognosis due to the activation of its noncanonical signaling pathway. Throughout cancer treatment, the emergence of drug-resistant tumor cells is relatively common. Since the early 2000s, researchers have focused on understanding the role of EphA2 in promoting drug resistance in different types of cancer, as well as finding efficient and secure EphA2 inhibitors. In this review, the current knowledge regarding induced resistance by EphA2 in cancer treatment is summarized, and the types of cancer that lead to the most cancer-related deaths are highlighted. Some EphA2 inhibitors were also investigated. Regardless of whether the cancer treatment has reached a drug-resistance stage in EphA2-overexpressing tumors, once EphA2 is involved in cancer progression and aggressiveness, targeting EphA2 is a promising therapeutic strategy, especially in combination with other target-drugs for synergistic effect. For that reason, monoclonal antibodies against EphA2 and inhibitors of this receptor should be investigated for efficacy and drug toxicity.

Endothelial Robo4 suppresses endothelial-to-mesenchymal transition induced by irradiation and improves hematopoietic reconstitution

Bone marrow ablation is routinely performed before hematopoietic stem cell transplantation (HSCT). Hematopoietic stem and progenitor cells (HSPCs) require a stable bone marrow microenvironment to expand and refill the peripheral blood cell pool after ablation. Roundabout guidance receptor 4 (Robo4) is a transmembrane protein exclusive to endothelial cells and is vital in preserving vascular integrity. Hence, the hypothesis is that Robo4 maintains the integrity of bone marrow endothelial cells following radiotherapy. We created an endothelial cell injury model with γ-radiation before Robo4 gene manipulation using lentiviral-mediated RNAi and gene overexpression techniques. We demonstrate that Robo4 and specific mesenchymal proteins (Fibronectin, Vimentin, αSma, and S100A4) are upregulated in endothelial cells exposed to irradiation (IR). We found that Robo4 depletion increases the expression of endoglin (CD105), an auxiliary receptor for the transforming growth factor (TGF-β) family of proteins, and promotes endothelial-to-mesenchymal transition (End-MT) through activation of both the canonical (Smad) and non-canonical (AKT/NF-κB) signaling pathways to facilitate Snail1 activation and its nuclear translocation. Endothelial Robo4 overexpression stimulates the expression of immunoglobulin-like adhesion molecules (ICAM-1 and VCAM-1) and alleviates irradiation-induced End-MT. Our coculture model showed that transcriptional downregulation of endothelial Robo4 reduces HSPC proliferation and increases HSC quiescence and apoptosis. However, Robo4 overexpression mitigated the damaged endothelium’s suppressive effects on HSC proliferation and differentiation. These findings indicate that by controlling End-MT, Robo4 preserves microvascular integrity after radiation preconditioning, protects endothelial function, and lessens the inhibitory effect of damaged endothelium on hematopoietic reconstitution.

HRD1-induced TMEM2 ubiquitination promotes ER stress-mediated apoptosis through a non-canonical pathway in intestinal ischemia/reperfusion

Intestinal ischemia/reperfusion (I/R) injury is a typical pathological course in the clinic with a high morbidity rate. Recent research has pointed out the critical role of ubiquitination during the occurrence and development of intestinal I/R by precisely mediating protein quality control and function. Here, we conducted an integrated multiomic analysis to identify critical ubiquitination-associated molecules in intestinal I/R and identified endoplasmic reticulum-located HRD1 as a candidate molecule. During intestinal I/R, excessive ER stress plays a central role by causing apoptotic pathway activation. In particular, we found that ER stress-mediated apoptosis was mitigated by HRD1 knockdown in intestinal I/R mice. Mechanistically, TMEM2 was identified as a new substrate of HRD1 in intestinal I/R by mass spectrometry analysis, which has a crucial role in attenuating apoptosis and promoting non-canonical ER stress resistance. A strong negative correlation was found between the protein levels of HRD1 and TMEM2 in human intestinal ischemia samples. Specifically, HRD1 interacted with the lysine 42 residue of TMEM2 and reduced its stabilization by K48-linked polyubiquitination. Furthermore, KEGG pathway analysis revealed that TMEM2 regulated ER stress-mediated apoptosis in association with the PI3k/Akt signaling pathway rather than canonical ER stress pathways. In summary, HRD1 regulates ER stress-mediated apoptosis through a non-canonical pathway by ubiquitinating TMEM2 and inhibiting PI3k/Akt activation during intestinal I/R. The current study shows that HRD1 is an intestinal I/R critical regulator and that targeting the HRD1/TMEM2 axis may be a promising therapeutic approach.

Connecting the ends: signaling via receptor tyrosine kinases and cytoskeletal degradation in neurodegeneration

Receptor tyrosine kinases (RTKs) are known to perform versatile roles in disease landscapes, which determine the fate of the cell. Although much has been discussed from the perspective of proliferation, this review focuses on the impact of RTK-mediated signaling and its role in cytoskeletal degradation, the penultimate stage of cellular degeneration. In the case of degenerative diseases such as Alzheimer’s disease (AD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), age-related macular degeneration (AMD), and type 2 diabetes mellitus (T2DM), RTK signaling has been reported to be perturbed in several studies. The implications of downstream signaling via these receptors through canonical and noncanonical pathways alter the status of actin filaments that provide structural integrity to cells. Degenerative signaling leads to the altered status of rat sarcoma (Ras), Ras homologous (Rho), Ras-related C3 botulinum toxin substrate (Rac), and cell division control protein 42 (Cdc42), the best-characterized components of the cytoskeleton remodeling machinery. RTKs, along with their diverse adaptor partners and other membrane receptors, affect the functionality of Rho family guanosine triphosphate hydrolases (GTPases), which are discussed in this review. To conclude, this review focuses on therapeutic strategies targeting RTKs and Rho GTPase-mediated pathways that can be more effective due to their combined multifactorial impact on neurodegenerative cascades.

Wings of Change: aPKC/FoxP-dependent plasticity in steering motor neurons underlies operant self-learning in Drosophila.

Motor learning is central to human existence, such as learning to speak or walk, sports moves, or rehabilitation after injury. Evidence suggests that all forms of motor learning share an evolutionarily conserved molecular plasticity pathway. Here, we present novel insights into the neural processes underlying operant self-learning, a form of motor learning in the fruit fly Drosophila. We operantly trained wild type and transgenic Drosophila fruit flies, tethered at the torque meter, in a motor learning task that required them to initiate and maintain turning maneuvers around their vertical body axis (yaw torque). We combined this behavioral experiment with transgenic peptide expression, CRISPR/Cas9-mediated, spatio-temporally controlled gene knock-out and confocal microscopy. We find that expression of atypical protein kinase C (aPKC) in direct wing steering motoneurons co-expressing the transcription factor FoxP is necessary for this type of motor learning and that aPKC likely acts via non-canonical pathways. We also found that it takes more than a week for CRISPR/Cas9-mediated knockout of FoxP in adult animals to impair motor learning, suggesting that adult FoxP expression is required for operant self-learning. Our experiments suggest that, for operant self-learning, a type of motor learning in Drosophila, co-expression of atypical protein kinase C (aPKC) and the transcription factor FoxP is necessary in direct wing steering motoneurons. Some of these neurons control the wing beat amplitude when generating optomotor responses, and we have discovered modulation of optomotor behavior after operant self-learning. We also discovered that aPKC likely acts via non-canonical pathways and that FoxP expression is also required in adult flies.

The origin and evolution of Wnt signalling.

The Wnt signal transduction pathway has essential roles in the formation of the primary body axis during development, cellular differentiation and tissue homeostasis. This animal-specific pathway has been studied extensively in contexts ranging from developmental biology to medicine for more than 40 years. Despite its physiological importance, an understanding of the evolutionary origin and primary function of Wnt signalling has begun to emerge only recently. Recent studies on very basal metazoan species have shown high levels of conservation of components of both canonical and non-canonical Wnt signalling pathways. Furthermore, some pathway proteins have been described also in non-animal species, suggesting that recruitment and functional adaptation of these factors has occurred in metazoans. In this Review, we summarize the current state of research regarding the evolutionary origin of Wnt signalling, its ancestral function and the characteristics of the primal Wnt ligand, with emphasis on the importance of genomic studies in various pre-metazoan and basal metazoan species.

The interplay between Wnt signaling pathways and microtubule dynamics.

Wnt signaling pathways represent an evolutionarily highly conserved, intricate network of molecular interactions that regulates various aspects of cellular behavior, including embryonic development and tissue homeostasis. Wnt signaling pathways share the β-catenin-dependent (canonical) and the multiple β-catenin-independent (non-canonical) pathways. These pathways collectively orchestrate a wide range of cellular processes through distinct mechanisms of action. Both the β-catenin-dependent and β-catenin-independent pathways are closely intertwined with microtubule dynamics, underscoring the complex crosstalk between Wnt signaling and the cellular cytoskeleton. This interplay involves several mechanisms, including how the components of Wnt signaling can influence the stability, organization, and distribution of microtubules. The modulation of microtubule dynamics by Wnt signaling plays a crucial role in coordinating cellular behaviors and responses to external signals. In this comprehensive review, we discussed the current understanding of how Wnt signaling and microtubule dynamics intersect in various aspects of cellular behavior. This study provides insights into our understanding of these crucial cellular processes.

Progression of retinal choroidal neovascularizationby latent human cytomegalovirus infection and immunological signaling among neonatal patients admitted to tertiary care hospital.

Choroidal neovascularization (CNV) is a serious condition that affects the retina, causing partial or complete blindness in people of different ages. While CNV is a common occurrence in various chorioretinopathies, research on its occurrence in neonates is limited. Human cytomegalovirus (HCMV) is a significant health threat to neonates, with a strong association with retinal angiogenesis. However, there has been limited investigation into HCMV-associated CNV progression. In this article, we extensively studied the expression of different inflammatory cytokines and chemokines during latent HCMV-associated retinal neovascularization. Our research found that HCMV-induced CNV progression was significantly prominent in the presence of AT2R-dependent angiogenesis (p < 0.001), whereas in the absence of HCMV, AT1R-dependent CCL-5-mediated angiogenesis was documented. We also observed significant increases in CCL-19, CCL-21 chemokine responses, followed by CCR-7 chemokine receptor activation (p < 0.001) in HCMV-induced CNV patients compared to HCMV non-induced CNV groups. Furthermore, significant changes in predictive chemokine markers of HCMV-induced CNV were positively correlated with HCMV viremia. These immunological alterations ultimately lead to the switching of NFκB canonical and noncanonical pathways, respectively, in HCMV-induced neonatal CNV and HCMV non-induced CNV. This clinical observation presents a novel hypothesis that ocular HCMV latency poses a noteworthy risk factor for the progression of retinal neovascularization through a distinctive immunological signaling pathway. The current study represents the first of its kind to report on this association, which may have significant implications for the clinical management of patients with ocular HCMV.

Canonical Wnt signaling pathway (WSP) alterations in metastatic prostate cancer.

221 Background: WSP aberrations have been implicated in the progression and treatment resistance of metastatic prostate cancer (mPCa). Sustained exposure of PCa to androgen deprivation has been shown to regulate the canonical WSP, induce beta-catenin expression and stabilization, and promote androgen independence. This study interrogates a large dataset to characterize the molecular and immune landscape of canonical WSP-altered mPCa and associations with survival outcomes. Methods: We examined PCa patients who underwent tissue-based DNA and RNA sequencing through a CLIA-certified commercial assay (Caris Life Sciences). Comprehensive genomic profiling included next-generation sequencing (NGS) of DNA (592-gene panel or whole exome) and RNA (whole transcriptome), on tissue samples from primary prostatic and/or metastatic sites. Tumor samples were categorized as having aberrant canonical Wnt signaling (WSP-act) if they harbored activating mutations in CTNNB, pathogenic RSPO2 fusions, or inactivating mutations in either APC or RNF43. Subset analysis was conducted in mPCa samples with microsatellite stability (MSS), after excluding those with RNF43 (p.G659fs*) mutations which are passenger events in MSI-high cancers. Statistical significance was determined using Fisher's Exact or X2 tests with Benjamini–Hochberg correction. Overall survival (OS) data was obtained from insurance claims and was analyzed using Kaplan-Meier estimation. Results: Of all PCa samples (4,150) (N = 4,150), 17.4% (n = 722) were classified as WSP-act; 299 (41.4%) were from the primary tumor and 423 (58.6%) were from metastatic sites. WSP-act tumors were more prevalent in brain (46%), liver (31%), lung (31%) and bone (25%) metastases compared to primary prostate cancers (10%). The MSS mPCa subgroup (N=1,638) comprised 321 WSP-act and 1,317 WSP-WT tumors. WSP-act mPCa exhibited more SPOP mutations (14.2% vs. 6.3%, p &lt; 0.001) as well as higher mRNA expression of CST1, NKD1, and ODAM (known canonical WSP activators) compared to WSP-WT cases. M2 macrophages predominated the tumor microenvironment, with a higher median fraction in WSP-act tumors compared to WSP-WT (7.2 vs. 6.1%, p &lt; 0.001). ROR1 gene expression was significantly elevated in WSP-act tumors from both primary and metastatic sites. There was no significant difference in OS outcomes between WSP-act and WSP-WT mPCa patients. Conclusions: WSP-act prostate cancer demonstrated a pronounced upregulation of ROR1 gene expression, underscoring its potential involvement in the crosstalk between the canonical and non-canonical Wnt signaling pathways. Additionally, the augmented levels of M2 macrophages in WSP-act tumors, combined with the reported role of ROR1 in tumor immunosuppression, suggests that ROR1 may contribute to immune evasion in WSP-act mPCa. Our findings may provide rationale for developing novel therapeutic strategies targeting Wnt-activated prostate cancers.

Matrix-bound nanovesicle-associated IL-33 supports functional recovery after skeletal muscle injury by initiating a pro-regenerative macrophage phenotypic transition

Injuries to skeletal muscle are among the most common injuries in civilian and military populations, accounting for nearly 60% of extremity injuries. The standard of care for severe extremity injury has been focused upon limb salvage procedures and the utilization of tissue grafts or orthotics in conjunction with rehabilitation to avoid amputation. Nonetheless, many patients have persistent strength and functional deficits that permanently impact their quality of life. Preclinical and clinical studies have shown that partial restoration of functional skeletal muscle tissue following injury can be achieved by the implantation of a biologic scaffold composed of extracellular matrix (ECM). These favorable outcomes are mediated, at least in part, through local immunomodulation. The mechanisms underlying this immunomodulatory effect, however, are poorly understood. The present study investigates a potential mechanistic driver of the immunomodulatory effects; specifically, the effect of selected ECM components upon inflammation resolution and repair. Results show that the host response to skeletal muscle injury is profoundly altered and functional recovery decreased in il33−/− mice compared to age- and sex-matched wildtype counterparts by 14 days post-injury. Results also show that IL-33, contained within matrix-bound nanovesicles (MBV), supports skeletal muscle regeneration by regulating local macrophage activation toward a pro-remodeling phenotype via canonical and non-canonical pathways to improve functional recovery from injury compared to untreated il33−/− counterparts. Taken together, these data suggest that MBV and their associated IL-33 cargo represent a novel homeostatic signaling mechanism that contributes to skeletal muscle repair.

Identification of a non-canonical chemokine-receptor pathway suppressing regulatory T cells to drive atherosclerosis

CCL17 is produced by conventional dendritic cells, signals through CCR4 on regulatory T (Treg) cells and drives atherosclerosis by suppressing Treg functions through yet undefined mechanisms. Here we show that conventional dendritic cells from CCL17-deficient mice display a pro-tolerogenic phenotype and transcriptome that is not phenocopied in mice lacking its cognate receptor CCR4. In the plasma of CCL17-deficient mice, CCL3 was the only decreased cytokine/chemokine. We found that CCL17 signaled through CCR8 as an alternate high-affinity receptor, which induced CCL3 expression and suppressed Treg functions in the absence of CCR4. Genetic ablation of CCL3 and CCR8 in CD4+ T cells reduced CCL3 secretion, boosted FoxP3+ Treg numbers and limited atherosclerosis. Conversely, CCL3 administration exacerbated atherosclerosis and restrained Treg differentiation. In symptomatic versus asymptomatic human carotid atheroma, CCL3 expression was increased, whereas FoxP3 expression was reduced. Together, we identified a non-canonical chemokine pathway whereby CCL17 interacts with CCR8 to yield a CCL3-dependent suppression of atheroprotective Treg cells.