Proximity Ligation Research Articles

Proteolysis of the pericellular matrix: Pinpointing the role and involvement of matrix metalloproteinases in early osteoarthritic remodeling

The pericellular matrix (PCM) serves a critical role in signal transduction and mechanoprotection in chondrocytes. Osteoarthritis (OA) leads to a gradual deterioration of the cartilage, marked by a shift in the spatial arrangement of chondrocytes from initially isolated strands to large cell clusters in end-stage degeneration. These changes coincide with progressive enzymatic breakdown of the PCM. This study aims to assess the role and involvement of specific matrix metalloproteinases (MMPs) in PCM degradation during OA.We selected cartilage samples from 148 OA patients based on the predominant spatial chondrocyte patterns. The presence of various MMPs (-1,-2,-3,-7,-8,-9,-10,-12,-13) was identified by multiplexed immunoassays. For each pattern and identified MMP, the levels and activation states (pro-form vs. active form) were measured by zymograms and western blots. The localization of these MMPs was determined using immunohistochemical labeling. To verify these results, healthy cartilage was exposed to purified MMPs, and the consecutive structural integrity of the PCM was analyzed through immunolabeling and proximity ligation assay. Screening showed elevated levels of MMP-1,-2,-3,-7, and -13, with their expression profile showing a clear dependency of the degeneration stage. MMP-2 and -7 were localized in the PCM, whereas MMP-1,-7, and -13 were predominantly intracellular. We found that MMP-2 and -3 directly disrupt collagen type VI, and MMP-3 and -7 destroy perlecan.MMP-2, -3, and -7 emerge as central players in early PCM degradation in OA. With the disease's initial stages already displaying elevated peaks in MMP expression, this insight may guide early targeted therapies to halt abnormal PCM remodeling. Statement of significanceOsteoarthritis (OA) causes a gradual deterioration of the articular cartilage, accompanied by a progressive breakdown of the pericellular matrix (PCM). The PCM's crucial function in protecting and transmitting signals within chondrocytes is impaired in OA. By studying 148 OA-patient cartilage samples, the involvement of matrix metalloproteinases (MMPs) in PCM breakdown was explored. Findings highlighted elevated levels of certain MMPs linked to different stages of degeneration. Notably, MMP-2, -3, and -7 were identified as potent contributors to early PCM degradation, disrupting key components like collagen type VI and perlecan. Understanding these MMPs' roles in initiating OA progression, especially in its early stages, provides insights into potential targets for interventions to preserve PCM integrity and potentially impeding OA advancement.

A beginner’s guide to immunohistochemistry

Using the affinity between antibodies and antigens, immunohistochemistry (IHC) is a helpful method for analysing the cellular components within tissue sections placed on glass slides. The chemistry of antibodies was not discovered until 1941, and since then, their use has increased due to the discovery of enzymes and fluorochromes conjugated to antibodies, enabling observations through optical or fluorescence microscopy (FM). Hence, immunofluorescence (IF), or IHC with fluorescent antibodies, has been associated with the advancement of FM and fluorochromes. Specifically, the discovery of three fluorochromes that emit blue, red and green led to a rise in IF application in the context of visualizing numerous components in a single cell. The proximity ligation assay (PLA) method, so termed because the antibody is conjugated with short DNA strands called PLA probes, is the most recent development in the field of antibody conjugation. Protein–protein interactions and sub-localization of proteins can both be studied using this technique. The improvement of conjugation techniques has occurred in parallel with the development of sophisticated microscopies, including confocal and slide scanners. Confocal microscopy allows for the reconstruction of three-dimensional structures while a slide scanner can digitally convert up to 100 slides' worth of high-resolution pictures. The first step in the IHC process is the collection and fixation of tissues from animal models and humans. Paraformaldehyde is the most commonly used fixative. Fixation prevents antigen blocking and creates cross-links between proteins to preserve tissue structures. Fixation is followed by cutting tissues using microtome or cryostat, then slides are treated for antigen exposition (antigen retrieval) and then included in Triton X solution, which increases membrane permeability. Subsequently, incubations with enzyme- or fluorochrome-conjugated antibodies enable observations via optical microscopy or FM, respectively. In clinical practice and research, IHC and IF are still helpful methods for identifying the antigen-proteins linked to specific diseases.

Myosin phosphatase targeting subunit1 controls localization and motility of Rab7-containing vesicles: Is myosin phosphatase a cytoplasmic dynein regulator?

Myosin phosphatase targeting subunit1 (MYPT1) is a critical subunit of myosin phosphatase (MP), which brings PP1Cδ phosphatase and its substrate together. We previously showed that MYPT1 depletion resulted in oblique chromatid segregation. Therefore, we hypothesized that MYPT1 may control microtubule-dependent motor activity. Dynein, a minus-end microtubule motor, is known to be involved in mitotic spindle assembly. We thus examined whether MYPT1 and dynein may interact. Proximity ligation assay and co-immunoprecipitation revealed that MYPT1 and dynein intermediate chain (DIC) were associated. We found that DIC phosphorylation is increased in MYPT1-depleted cells in vivo, and that MP was able to dephosphorylate DIC in vitro. MYPT1 depletion also altered the localization and motility of Rab7-containing vesicles. MYPT1-depletion dispersed the perinuclear Rab7 localization to the peripheral in interphase cells. The dispersed Rab7 localization was rescued by microinjection of a constitutively active, truncated MYPT1 mutant, supporting that MP is responsible for the altered Rab7 localization. Analyses of Rab7 vesicle trafficking also revealed that minus-end transport was reduced in MYPT1-depleted cells. These results suggest an unexpected role of MP: MP controls dynein activity in both mitotic and interphase cells, possibly by dephosphorylating dynein subunits including DIC.

Abstract PO4-28-08: Unraveling the Role of BRCA1-BARD1 E3 Ubiquitin Ligase in DNA Repair: A Promise for Enhanced Chemotherapy Outcomes

Abstract BACKGROUND: BRCA1, a central player in gynecological cancers, collaborates with BARD1 to shape a potent complex pivotal for DNA binding and ubiquitin E3 ligase activities. This complex impacts a myriad of biological pathways. Notably, BRCA1-BARD1’s role in tumor suppression and homology-directed DNA repair (HDR) has been spotlighted. Our innovative approach used RING-domain mutations to craft ligase-dead BRCA1-BARD1 mutants, hypothesizing that these mutants would offer fresh insights into the DNA repair dynamics of BRCA1-BARD1. METHODS: Full length BRCA1-BARD1 or truncated mutants, and histones were purified from E. coli. or insect cells. Nucleosomes were assembled for in vitro ubiquitylation reaction and binding assays. Stable mammalian cell lines HeLa and MDA-MB-436 that express wild type or mutant forms of BRCA1 and BARD1 were established for a host of analyses, including cellular fractionation, foci analysis, Proximity ligation assay (PLA), comet assays and clonogenic survival assays with various DNA damage agents. RESULTS: By systematic biochemical screening and a series of in vitro assays, we generated a truly BRCA1-BARD1 E3 dead mutant, BRCA1I26A, L63A, K65A-BARD1 (BRCA1-E3d), which lacks E3 ligase activity but possesses all other known attributes such as retention of BRCA1-BARD1 heterodimeric structure formation, DNA binding, and intact RAD51-mediated recombinase activity. To our surprise, we discovered that previously described BRCA1-BARD1 RING-domain mutant (BRCA1-I26A) still possessed ubiquitylation activity not found in BRCA1-E3d. To determine the biological significance of these mutants, cells stably expressing BRCA1-E3d were treated with various DNA-damaging agents and shown to more sensitive than WT or previously identified mutants. DNA repair pathway reporter assays determined these cells were deficient in various repair pathways compared to their BRCA1-WT counterparts. Further studies demonstrate that BRCA1-BARD1 E3 ligase is required for DNA resection during HDR, as evidenced in reduced levels of DNA repair-related foci formation such as RPA, RAD51 and CtIP in BRCA1-E3d cells. Furthermore, compared to BRCA1-WT cells, BRCA1-E3d cells were more sensitive to DNA damage reagents after depletion of 53BP1, which indicates BRCA1-BARD1 E3 ligase function also contributes to later stages of DNA repair completion. CONCLUSIONS: Our work dispels prevailing ambiguities surrounding BRCA1-BARD1 E3 ligase functions, underscoring its paramountcy in genome repair. This trailblazing research not only enriches our understanding but also beckons therapeutic interventions targeting tumor suppression. The unveiling of BRCA1-BARD1 E3 ligase's intricate regulatory dynamics combined with our novel mutants paves the way for an exciting new era in cancer therapeutics, hinting at superior treatments to enhance patient recovery. Citation Format: Wenjing Li, Meiling Wang, Nozomi Tomimatsu, Jae-Hoon Ji, Salvador Alejo, Gangadhara R. Sareddy, Sandeep Burma, Rachel Klevit, Weixing Zhao. Unraveling the Role of BRCA1-BARD1 E3 Ubiquitin Ligase in DNA Repair: A Promise for Enhanced Chemotherapy Outcomes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-08.

Abstract PO4-24-03: Adiponectin modulates cell polarity according to ERα expression in breast cancer cells

Abstract INTRODUCTION: Tumor microenvironment (TME) is a complex and heterogeneous network, consisting of stromal and immune cells, embedded with cytokines, growth factors, soluble receptors, and exosomes. Mutual and dynamic crosstalk among cancer, stromal and immune cells predispose cancer cells to metastasis. Particularly, the main component of breast TME is represented by adipocytes, which secretory activity is compromised in obesity. Indeed, hypertrophic, and hyperplastic adipocytes undergo to a modified cytokines secretory pattern, characterized by a reduced secretion of Adiponectin, now recognized as a crucial factor in the pathogenesis of breast cancer. LKB1 is one the most important effector of Adiponectin transduction pathways, and recently it has been demonstrated its role as ERα coactivator in promoting breast cancer cell growth. Moreover, LKB1 is a key regulator in controlling cell polarity along with E-cadherin and Cdc42. Cell polarity has been considered essential for mammary epithelial integrity, and disruption of individual polarity proteins is sufficient to induce tumor progression. The aim of the present study was to investigate the effects of adiponectin on the regulation and alteration of cell polarity, essential for breast cancer cell growth and invasiveness. METHODS: Immunofluorescence analyses were done to detect protein localization in breast cancer cells. Protein expression was performed by immunoblotting in the presence or absence of ERa and LKB1 siRNA. LKB1/Cdc42 and LKB1/E-cadherin interaction was detected by Proximity ligation and immunoprecipitation assay respectively. Wound healing assay was carried out to evaluate GM130 orientation. Growth and metastatic potential of breast cancer cells were assessed in vivo by orthotopic model and metastatic study respectively. RESULTS: In Adiponectin-treated MCF-7 cells LKB1 is mostly present in the nucleus, recruited as ERa-coactivator, while in BT20 cells LKB1 is localized with E-cadherin in the cytosolic compartment, suggesting its role in polarity. Our results showed that in MCF-7 cells Adiponectin increases Cdc42 level depending on LKB1 expression. Moreover, Cdc42 and LKB1 interact and colocalized in the nucleus of Adiponectin-treated MCF-7 cells. On the contrary, LKB1 and Cdc42 are located with E-cadherin in the cytosol of BT20 cells, addressing their cytosolic cooperation in maintaining cell polarity, as confirmed by an increased Cdc42 activity. Immunofluorescence analyses demonstrated a lack of Golgi realignment toward the front of the leading edge in MCF-7 cells treated with Adiponectin, not observed in BT20 cells. All this suggest a loss of cell polarity organization that predispose to cancer growth and progression, as confirmed by an increased tumor volume mass and a major number of lung metastatic events upon tail vein injection of adiponectin-treated ERα-positive cells with respect to untreated cells. CONCLUSION: In ERα-positive breast cancer cells Adiponectin modulates the expression of the main proteins involved in cell polarity, leading to increased growth, progression, and distant metastasis. Citation Format: Giuseppina Daniela Naimo, Martina Forestiero, Alessandro Paolì, Luca Gelsomino, Rocco Malivindi, Adele Elisabetta Leonetti, Francesca Giordano, Maria Luisa Panno, Loredana Mauro, Sebastiano Andò. Adiponectin modulates cell polarity according to ERα expression in breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-03.

Elucidating the Role of SYK in the Intestinal Epithelium

Background: Inflammatory Bowel Disease (IBD) involves chronic and recurring inflammation of the gastrointestinal tract. We identified several patients presenting with immune dysregulation and systemic inflammation, including inflammation of the gut. Analysis of Whole Exome Sequencing data from pediatric IBD patients has identified five rare protein coding Spleen Tyrosine Kinase (SYK) variants. Functional studies have demonstrated increased kinase activity implicating them in disease pathophysiology. While the role of SYK is well characterized in immune cells, it is not understood what role it plays in the intestinal epithelium. Aims: The primary aim of this project is to understand the role SYK plays in the intestinal epithelium, what molecular pathways it signals through, and how its dysregulation leads to intestinal disease. Methods: To understand SYKs role in the intestinal epithelium, a BioID screen was used to identify novel SYK interacting proteins. BioID was done with wild type SYK as well as the patient derived variants in HEK293 cells. BioID hits were validated using co-immunoprecipitation (CoIP) and proximity ligation assay (PLA). Further functional analysis was done in Caco2 cells, an intestinal epithelial cell model, with western blot, ELISA, and luciferase assays to assess the effect of SYK activity on downstream inflammatory pathways. Finally, we investigated the effect of small molecule kinase inhibitors to explore possible novel treatment options. Results: The BioID screen revealed several high confidence interactors, with dysregulated interactions in the patient variants. USP25, a ubiquitin specific peptidase that is widely expressed, including in the intestinal epithelium, was identified as one of the strongest hits based on peptide counts. The interaction was validated with CoIP and PLA, and further analysis showed SYK activation decreased USP25 protein levels by up to eight-fold. The secretion of the proinflammatory chemokine, IL-8, in response to lipopolysaccharide (LPS) was found to be almost twice as high in Caco2 cells with patient SYK variants relative to WT. Cells expressing the patient variants of SYK showed increased NFkB and AP1 levels, indicating increased inflammatory gene expression when stimulated with 100ng/mL LPS. NFkB was also active for longer in cells expressing the patient variant of SYK post stimulation, with WT cells returning to baseline in under half an hour, while the patient variant stayed in an inflamed state for 36 to 48 hours after LPS was removed. Conclusions: Increased SYK activity is associated with the degradation of USP25. USP25 is known to cleave ubiquitin off TNF receptor associated factors (TRAFs). TRAFs are activated downstream of innate immune receptors such as TLR4, and need to be ubiquitinated for activation. USP25, which is responsible for the deubiquitination of these TRAFs, acts as a negative regulator of these pathways. The hyperactive SYK variants degrade USP25 and impair the cells’ ability to switch off inflammatory signaling pathways, indicated by the cells elongated period in an inflamed state. This research is funded by grants from Canadian Institute for Health Research (CIHR) and the Leona M. and Harry B. Helmsley Charitable Trust. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The assembly factor Dmxl1 is required for H+(V)-ATPase assembly and function in mammals in vivo

The V-ATPase (proton pump) generates electrochemical gradients across cell membranes, driving essential biological processes. Critically, the two V-ATPase domains, V1 and VO, must assemble to produce a functional holoenzyme. V-ATPase dysfunction results in serious pathologies such as cancer, neurodegeneration, and diabetes, as well as systemic acidosis caused by reduced activity of proton-secreting intercalated cells (IC) in the kidney. However, very little is known about the molecular regulation of V-ATPase in mammals. We have identified a novel interactor of the IC-enriched B1 subunit of the mammalian V-ATPase, Drosophila melanogaster X chromosomal gene-like 1 protein (Dmxl1), aka Rabconnectin-3A (Rbcn3A). The yeast homologue of Dmxl1, Rav1p, is part of a protein complex that catalyzes the reversible assembly of the V1 and VO domains, thus regulating proton pumping activity. We, therefore, hypothesized that Dmxl1 is an assembly factor for the V-ATPase in mammalian cells. We generated kidney IC-specific Dmxl1 knockout (KO) mice and they had a high urine pH, similar to B1 V-ATPase KO mice, suggesting impaired V-ATPase function. Western blotting of kidney lysates showed decreased B1 V-ATPase expression, but other V-ATPase subunits were normal. B1 (V1 domain) and a4 (VO domain) subunits were immunolocalized in ICs and apical localization quantified using FIJI. Both B1 and a4 were more intracellular in Dmxl1 KO ICs relative to control mice. A Pearson’s correlation analysis of B1 and a4 staining revealed a significant decrease in colocalization in Dmxl1 KO ICs, suggesting reduced B1/a4 assembly. In parallel, subcellular fractionation experiments revealed less V1 domain-associated B1 subunit in the membrane fraction relative to the cytosol. Furthermore, a Proximity Ligation Assay (PLA) was performed using probes against B1 and a4 to confirm decreased assembly of the two domains in KO ICs. When the two probes are within 40 μM of each other, like when the domains are assembled, this results in a fluorescent PLA punctum. As expected, we observed a significant decrease in the area of PLA puncta per cell in KO animals relative to control animals. These data demonstrate that the V-ATPase is less assembled in Dmxl1 KO ICs, relative to controls. We propose that loss of Dmxl1 reduces assembly of the V-ATPase holoenzyme, thereby inhibiting its proton pumping function. Dmxl1 may mediate recruitment of the V1 domain to the membrane and facilitate its assembly with the transmembrane VO domain. In the absence of Dmxl1, V1 is instead targeted for degradation, explaining the inability of these mice to properly acidify their urine. We conclude that Dmxl1 is a bona-fide mammalian V-ATPase assembly factor that functions similarly to its yeast homolog, Rav1p in affecting V-ATPase assembly. NIH/NIDDK R01DK121848 to DB. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1046: Miro1 Promotes Mitochondria ER Contact Sites Formation In Vascular Smooth Muscle Cells During G1/s

Vascular smooth muscle cell (VSMC) proliferation plays a key role in vascular disorders including atherosclerosis. Mitochondria promote VSMC proliferation by mitochondrial Ca 2+ regulation, ROS and ATP production. Mitochondria-ER contact sites (MERCS) promote signal transduction to mitochondria. MIRO1, an outer mitochondrial membrane GTPase, drives intracellular mitochondrial mobility. In drosophila, MIRO1 controls MERCS formation. However, it is unknown whether this interaction promotes key cellular eukaryotic phenotypes, such as proliferation. Here, we tested whether MIRO1 promotes VSMC proliferation by modulating MERCS and mitochondrial Ca 2+ entry. MIRO1 deletion decreased neointima formation in vivo, and abolished VSMC proliferation and G1/S transition in vitro. In WT VSMCs, proximity ligation assays (PLA) and split-GFP imaging revealed that MERCS formation increased in G1/S phase along with recruitment of MIRO1. MIRO1 deletion abolished cell cycle-dependent regulation of MERCS while increasing the expression of MERCS proteins GRP75 and IP3R. MIRO1 deletion also blocked increases in baseline mitochondrial [Ca 2+ ] by mitoPericam imaging and decreases in ER [Ca 2+ ] by CEPIA-ER in G1/S phase. Additionally, the mitochondrial membrane potential (TMRM) and mitochondrial respiration significantly increased at G1/S in WT VSMCs but not in MIRO1-/- cells. In WT VSMCs, pyruvate dehydrogenase activity increased G1/S phase while cytosolic ATP levels (ATP sensor) were increased. In MIRO1-/- cells, both parameters did not change. These data demonstrate that MERCS formation and mitochondrial [Ca 2+ ] increases during the cell cycle and is controlled by MIRO1. Furthermore, MIRO1 deletion decreases cellular ATP levels in VSMCs. In conclusion, we propose that MIRO1 controls MERCS and increased mitochondrial [Ca 2+ ] that drives metabolic activity and proliferation.

Nogo-B modulates physiological and pathological angiogenesis during development and disease

Angiogenesis, i.e., the formation of new blood vessels, is essential for normal development and functional vessel formation in ischemic diseases. During angiogenesis, endothelial cells (ECs) coordinate migration, proliferation, and metabolic fluxes to adapt to branching vessels' increasing energy demands. Nogo-B is a ubiquitously expressed reticulon family protein mainly localized to the endoplasmic reticulum (ER) membrane. Our previous studies have shown that the genetic loss of Nogo-B results in aberrant vascular remodeling and dysregulated endothelial barrier function. The current study aims to examine the role of endothelial Nogo-B in regulating developmental angiogenesis and pathological angiogenesis in retinopathy of prematurity (ROP). We hypothesized that endothelial Nogo-B is required for physiological and pathological angiogenesis, and Nogo-B depletion attenuates pathological angiogenesis. Using tamoxifen-inducible, endothelial cell-specific Nogo-B−/− (Nogo-BiECKO) mice, Nogo-B global knockout mice, and C57Bl/6 (WT) mice, our results show that loss of Nogo-B in ECs significantly delayed Isolectin B4 (IB4)-labeled retinal blood vessel sprouting. Impaired EC proliferation was observed by the staining of and incorporation of 5-ethynyl-2′-deoxyuridine (EdU). Furthermore, using the Oxygen Induced Retinopathy (OIR) model to mimic ROP, we could demonstrate that loss of Nogo-B in EC resulted in reduced neovascularization compared to WT. Mechanistically, using Percoll gradients, ultra-resolution microscopy, proximity ligation assay, and electron-microscopy, we show that Nogo-B is localized in the mitochondria-associated ER membrane (MAM) and regulated MAM remodeling. Using seahorse assay, quantitative RT-PCR, and metabolomics analysis, we determined that loss of Nogo-B altered VEGF-mediated EC glycolysis, which is needed for the energy demand during angiogenesis. These data suggest that Nogo-B regulates glucose metabolism in EC and plays a pivotal role in physiological and pathological angiogenesis. Targeting endothelial Nogo-B may provide a plausible approach to controlling the formation of pathological neovascularization in retinal diseases, such as ROP, diabetic retinopathy, and wet forms of age-related macular degeneration. This work was supported by the National Institutes of Health (R01HL153599) and awards from the American Heart Association award to JY. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2122: A Multiomic Timecourse Atlas Of Murine Vascular Disease Smooth Muscle Cell Transition Phenotypes And Their Perturbation With Tcf21 Knockout

Background: Vascular smooth muscle cells (SMC) contribute to heritable CAD risk and undergo cell changes to fibroblast-like (FMC) and osteochondrogenic (CMC) phenotypes. We perform molecular characterization of this transition process over the course of atherosclerosis development to better understand how causal SMC CAD genes such as TCF21 drive the phenotypic transition process to alter CAD risk. Methods/Results: Deep multiomic profiling of aortic roots from control atherosclerosis model ( Myh11Cre ERT2 , ROSA tdT/+ , ApoE -/- ) mice with SMC specific lineage tracing on high fat diet were harvested for 7 scRNA and 6 scATAC time points across 16 weeks. Similarly, aortic roots from Tcf21 knockout ( Myh11Cre ERT2 , Tcf21 ΔSMC/ΔSMC , ROSA tdT/+ , ApoE -/- ) were collected at 3 timepoints for paired scRNA/scATAC. Waddington-Optimal Transport (WOT) mapped transcriptional trajectories across SMC lineage clusters which terminate in FMC and CMC phenotype clusters. PANDO/CellOracle frameworks were used to create gene regulatory networks with the ability to model SMC transition perturbations in silico . These analyses generated a multiomic timecourse atlas demonstrating the lineage traced phenotypic progression of SMC to FMC and CMC. Upon knockout of Tcf21 , we discern notable shifts in the SMC to FMC transition characterized by differential scRNA transcriptional pathways and scATAC shifts in transcription factor (TF) motif accessibility which also overlap with WOT predicted TF enrichment for each cell state. These integrative analyses revealed Tcf21 regulation of Cebpb and the Tead1 -Hippo pathways as novel mechanisms for mediating its role in modulation of disease related processes such as inflammation, response to TGF- β signaling and cell proliferation. Further mechanistic validation is performed through a combination of TF binding site analysis with TEAD1 and CEBPB ChIP-Seq in human coronary artery SMCs, dual luciferase reporter assays and proximity ligation assays. Conclusion: We construct a single cell atherosclerosis timecourse to model mouse SMC cell states and perturb CAD gene Tcf21 to elucidate the interplay between epigenetic and transcriptional mechanisms which affect key SMC transition elements such as CEBPB and the TEAD1 -Hippo axis.

Induction of CXCL3 in airway epithelial cells by a novel IL-9 receptor complex

Abstract Cytokine signaling is critical for cell-to-cell communication in development, homeostasis and disease. Interleukin (IL)-9 signaling promotes allergic diseases by acting on both immune and structural cells. In hematopoietic cells, IL-9 receptor (IL-9R) consists of two subunits – the α-chain (IL-9Rα) and a common γ (γc) chain. However, IL-9-responsive epithelial cells express IL-9Rα but not the γc chain raising a question on how these cells respond to IL-9 in the absence of the established IL-9R complex. Here, we discovered that in airway epithelial cells, IL-9Rα pairs with IL-13Rα1, forming a novel type II IL-9R. Using proximity ligation assay, we show that the IL-9Rα /IL-13Rα1 chains are in IL-9-dependent proximity in both mouse tracheal epithelial cells (mTEC) and human small airway epithelial cells (SAEC). IL-9-induced STAT3 activation in SAEC was blocked by anti-IL-13Rα1 but not by anti-γc. Using RNA-seq, in vitro IL-9 treatment of SAEC induced the expression of genes including CXCL3, a key chemokine implicated in asthma. Importantly, intranasal administration of IL-9 increased Cxcl3 expression in mTECs and CXCL3 protein in the lung, that corresponds with an expansion in the number of CXCR2+ immune cells in the lung. Thus, we identify a novel type II IL-9R which is composed of IL-9Rα and IL-13Rα1 and show that IL-9 signaling via type II IL-9R induces CXCL3 expression in epithelial cells to contribute to airway inflammation.

Abstract 2140: The Tgfb-Whsc1 Axis Drives Macrophage To Myofibroblast Transition During Wound Repair

During the proliferative phase of tissue repair macrophages can transition to myofibroblasts, which promote wound contraction and closure. The mechanisms that control this transition remain unknown and are not well studied. Here, using a combination of murine transgenic models of disease, loss of function approaches, pharmacologic inhibition in vivo, and single cell RNA sequencing (scRNA-seq) in human wounds, we identified that a histone methyltransferase, Whsc1, regulates the fibrotic gene program in macrophages during wound repair. Specifically, we identified that Whsc1 was upregulated in wound macrophages (CD3 - CD19 - NK1.1 - Ly6G - CD11b + ) in response to TGFβ. Genetic loss of TGFβ-mediated signaling as well as siRNA knockdown of Whsc1 in bone marrow derived macrophages (BMDMs) decreased TGFβ-dependent expression of Acta2, Col1a1, Col3a1 (p<0.05). Further, TGFβ stimulation resulted in increased Whsc1 and H3K36 dimethylation at fibrotic gene promoters in wound macrophages (p<0.05). Importantly, local pharmacologic inhibition of Whsc1 in vivo significantly impaired wound healing, suggesting that Whsc1 is critical for normal wound repair. We next examined the role of Whsc1 in pathologic healing conditions (i.e. diabetes) where the macrophage-myofibroblast transition is impaired. Whsc1 expression was decreased in human macrophages analyzed by scRNA-seq of diabetic wounds and in murine wound macrophages from a diabetic model (DIO) (p<0.05). Interestingly, in DIO BMDMs, Whsc1 did not traffic to fibrotic promoters after TGFβ treatment, but rather remained at NFkB-regulated inflammatory gene promoters ( Il1b, Il6, Tnf ). On further examination of this, using co-immunoprecipitation and proximity ligation assay in BMDMs, we demonstrated increased physical interaction between RelA and Whsc1 in diabetic macrophages. Knockdown of RelA in DIO macrophages increased Whsc1 enrichment at fibrotic promoters, suggesting RelA sequestered Whsc1 at inflammatory gene promoters (p<0.05). The identification of TGFβ-Whsc1 as a driver of macrophage-myofibroblast transition in normal and pathological tissues allows for development of novel therapies to improve tissue repair, and targeting RelA in macrophages may improve diabetic wound healing.

Pressure regulation of L-type Ca2+ channels and its role in myogenic response

L-type calcium (Ca2+) channels hold a pivotal position in governing intracellular Ca2+ concentration within smooth muscle and the establishment of myogenic tone. Their primary regulatory mechanism has historically been linked to voltage. Nevertheless, it remains a subject of inquiry whether voltage represents the exclusive modulator or whether these channels also exhibit mechanosensitivity. This study seeks to elucidate the influence of pressure stimuli on L-type Ca2+ channels in the resistance arteries of rodents (rats and mice). The investigative approach spans from the scrutiny of individual smooth muscle cells employing methodologies such as whole-cell and cell-attached patch-clamp electrophysiology, immunohistochemistry, proximity ligation assay, and super-resolution microscopy to the evaluation of intact arteries utilizing pressure myography. The findings obtained through whole-cell patch-clamp electrophysiology unveil a notable augmentation in L-type Ca2+ current in response to pressure stimuli. Subsequent scrutiny at the level of single-channel activity discloses that this augmentation is ascribed to an enhancement in functional coupling. Further inquiry reveals that the intensified functional coupling in response to pressure is contingent upon two principal factors: 1) the facilitation of cooperative gating mediated by protein kinase C (PKC), and 2) the traffcking of subunits to caveolae. Notably, functional experiments, involving depolarization elicited through the application of 30 mM KCl, underscore that arterial tone and cytosolic Ca2+ concentration responses are markedly more pronounced when the arteries are subjected to a pressure of 80 mmHg, as opposed to 20 mmHg. The culmination of these observations from both patch-clamp experiments and functional investigations supports the assertion that L-type Ca2+ channels are indeed amenable to mechanosensory influences, in addition to their well-documented voltage-dependent behavior. In summation, this investigation underscores that the regulatory landscape of L-type Ca2+ channels transcends the confines of voltage control, emphasizing the significance of considering pressure sensitivity in both physiological and pathological contexts. Ongoing research endeavors aim to delineate with precision the signaling complexes within vascular smooth muscle that respond to mechanical forces. Supported by the Canadian Institute for Health Research, National Institute of Health and the Rorabeck Chair in Vascular Biology and Neuroscience. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dual specificity phosphatase 12 deficiency reveals its role in maintaining T cell survival

Abstract Dual specificity phosphatases (DUSPs) are protein phosphatases that regulate cellular signaling pathways by dephosphorylating threonine/serine and tyrosine residues on the substrates. DUSP12 mediates the regulation of Toll-like receptor signaling, cell cycle, hepatocyte metabolism, cardiac hypertrophy, and fibrosis. Moreover, a nonsynonymous mutation of DUSP12 has been identified in patients with T-cell-mediated autoimmune diseases. However, the regulatory roles and mechanisms of DUSP12 in T cells remain unclear. In this study, we investigated the functional regulation and molecular mechanism of DUSP12 in T cells using T cell-specific DUSP12 conditional knockout mice. We found that DUSP12 cKO mice had severe T-cell lymphopenia. More interestingly, DUSP12 cKO mice spontaneously developed systemic inflammation including splenomegaly and intestinal inflammation. Furthermore, our results showed that DUSP12 localized to mitochondria and was essential for mitochondria integrity and cellular oxidative stress in T cells. Using mass spectrometry analysis, western blotting, immunofluorescent staining, and proximity ligation assay, we identified a novel DUSP12-interacted protein in T cells. The functional regulation of DUSP12 on this novel protein in T cells was also explored. Our current results made a significant breakthrough in DUSP12 functions in T cells. Also, DUSP12 may serve as a potential biomarker and therapeutic target for inflammatory disease.

Differential Downregulation of Subcellular Specific adrenoceptor Signaling in The Heart

Objective: Recent studies have shown that intracellular b1- adrenoceptors (b1ARs) are associated with the (Sarco)endoplasmic reticulum calcium ATPase 2a (SERCA2a) on the sarcoplasmic reticulum (SR) membrane. However, the mechanism of how this plasma membrane- and SR-associated pool of b1ARs signals downregulation under chronic sympathetic stimulation is not well understood. We aim to study these subcellular β1AR signals in different cellular compartments in a heart failure (HF) mouse model with chronic infusion of isoproterenol (ISO). Results and Methods: We investigated the association of b1AR with L-type calcium channel (LTCC), ryanodine receptor 2 (RyR2), and SERCA2a by utilizing proximity ligation assay (PLA) and confocal imaging in HF induced by chronic ISO infusion (30 mg/kg/day, 2 weeks). Chronic ISO infusion resulted in a downregulation of b1AR, and decreased receptor association with LTCC and RyR2 at the tubular membrane. In comparison, the b1AR association with SERCA2a was increased; however, the local cAMP-PKA mediated phospholamban phosphorylation is downregulated. Using Förster Resonance Energy Transfer (FRET) with genetically encoded biosensors to probe subcellular β1AR-PKA signaling, we observed reduced local ISO-induced PKA signaling at the RyR2 but not the SERCA2a nanodomains. Meanwhile, the expression of phosphodiesterases (PDEs) was altered including the downregulation of PDE4D and PDE3A and the upregulation of PDE2A, PDE4A, and PDE4B, which alters the local PKA activities in individual subcellular nanodomains. Interestingly, the localized PKA signal induced by endogenous norepinephrine at the SERCA2a nanodomain was impaired. Inhibition of monoamine oxidase A rescued the norepinephrine-induced local PKA signaling at the SERCA2a nanodomain and myocyte sarcomere shortening. Conclusion: We have uncovered distinct remodeling of b1AR signaling at the RyR2 and SERCA2a nanodomains in mouse HF after a chronic infusion of ISO. Our study offers strategies to restore the subcellular nanodomain cardiac b1AR signaling and contractile function in HF associated with chronic adrenergic stress, pointing toward potential therapeutic targets. National Institutes of Health grants R01-HL147263 and HL162825, Veteran Affair Merit grants IK6BX005753, 01BX002900 and BX005100. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Enzymatic Function of Kvβ2 Controls Vascular Potassium Channel Activity

Voltage-gated potassium (Kv) channels expressed in vascular smooth muscle control membrane potential and thereby regulate vascular tone and organ perfusion. Native Kv1 channels in the resistance vasculature consist of an alpha pore domain in complex with intracellular beta (Kvβ) proteins, which are active aldo-keto reductases (AKR6A). Recent work suggests that the AKR function of Kvβ2 is required for vascular Kv1 channel NAD(P)(H) redox sensing and O2 sensitivity of resistance arterial tone. Nonetheless, whether acute changes in Kvβ2 AKR function impact vascular Kv1 activity is unknown. We propose that Kv1 gating is sensitive to changes in the levels of endogenous substrates and post-translational modification of Kvβ2. Here, we tested the hypothesis that the availability of the glycolytic byproduct and purported Kvβ substrate, methylglyoxal, and protein kinase C-dependent phosphorylation differentially regulate vascular Kvβ2 function. Using the inside-out configuration of the patch clamp technique, we measured the open probability (nPo) of Kv channels present in patches excised from human coronary smooth muscle cells. Perfusion of methylglyoxal (5 μM) resulted in significantly increased Kv channel nPo (3.3x10−5 ± 9.5x10−6 to 3.2x10−4 ± 2.1x10−4; n=5) and this effect was enhanced (~8-fold) in the presence of 100 μM NADPH, suggesting that Kv sensitivity to methylglyoxal is cofactor-dependent. Based on this, we next tested whether Kvβ phosphorylation regulates AKR catalysis and influences vascular Kv1 redox sensitivity. Using in situ proximity ligation, we found that serine phosphorylation of Kvβ2 in smooth muscle was increased upon treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA; 100 nM), indicating that smooth muscle Kvβ2 is a phosphorylation target under these conditions. In vitro phosphorylation of purified Kvβ2 with PKCα significantly slowed catalysis (11.3 ± 0.3 vs. 6.5 ± 0.2 μmoles/min/mg protein, control and PKCα-treated rat Kvβ2.1, respectively). Pretreatment of cells with 10 nM PMA before excising inside-out membrane patches eliminated the increase in Kv channel nP(o) evoked by methylglyoxal (±NADPH). Moreover, ex vivo treatment of isolated coronary arteries with PMA (10 nM) abolished vasodilation in response to elevated NADH:NAD+ via application of 2–5 mM external L-lactate. Together, these results suggest that vascular smooth muscle Kv1 channels are functionally regulated by endogenous substrates and phosphorylation of intracellular Kvβ proteins. We propose that targeting vascular Kvβ AKR function may represent a novel strategy to modify K+ channel redox sensitivity and thus strengthen the coupling between metabolic demand and organ perfusion. T32-ES011564 and R01HL163818. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Paxillin regulates androgen receptor expression associated with granulosa cell focal adhesions.

Paxillin is a ubiquitously expressed adaptor protein integral to focal adhesions, cell motility, and apoptosis. Paxillin has also recently been implicated as a mediator of nongenomic androgen receptor (AR) signaling in prostate cancer and other cells. We sought to investigate the relationship between paxillin and AR in granulosa cells (GCs), where androgen actions, apoptosis, and focal adhesions are of known importance, but where the role of paxillin is understudied. We recently showed that paxillin knockout in mouse GCs increases fertility in older mice. Here, we demonstrate that paxillin knockdown in human granulosa-derived KGN cells, as well as knockout in mouse primary GCs, results in reduced AR protein but not reduced mRNA expression. Further, we find that both AR protein and mRNA half-lives are reduced by approximately one-third in the absence of paxillin, but that cells adapt to chronic loss of paxillin by upregulating AR gene expression. Using co-immunofluorescence and proximity ligation assays, we show that paxillin and AR co-localize at the plasma membrane in GCs in a focal adhesion kinase-dependent way, and that disruption of focal adhesions leads to reduced AR protein level. Our findings suggest that paxillin recruits AR to the GC membrane, where it may be sequestered from proteasomal degradation and poised for nongenomic signaling, as reported in other tissues. To investigate the physiological significance of this in disorders of androgen excess, we tested the effect of GC-specific paxillin knockout in a mouse model of polycystic ovary syndrome (PCOS) induced by chronic postnatal dihydrotestosterone (DHT) exposure. While none of the control mice had estrous cycles, 33% of paxillin knockout mice were cycling, indicating that paxillin deletion may offer partial protection from the negative effects of androgen excess by reducing AR expression. Paxillin-knockout GCs from mice with DHT-induced PCOS also produced more estradiol than GCs from littermate controls. Thus, paxillin may be a novel target in the management of androgen-related disorders in women, such as PCOS.

Membrane Heteroreceptor Complexes as Second-Order Protein Modulators: A Novel Integrative Mechanism through Allosteric Receptor-Receptor Interactions.

Bioluminescence and fluorescence resonance energy transfer (BRET and FRET) together with the proximity ligation method revealed the existence of G-protein-coupled receptors, Ionotropic and Receptor tyrosine kinase heterocomplexes, e.g., A2AR-D2R, GABAA-D5R, and FGFR1-5-HT1AR heterocomplexes. Molecular integration takes place through allosteric receptor-receptor interactions in heteroreceptor complexes of synaptic and extra-synaptic regions. It involves the modulation of receptor protomer recognition, signaling and trafficking, as well as the modulation of behavioral responses. Allosteric receptor-receptor interactions in hetero-complexes give rise to concepts like meta-modulation and protein modulation. The introduction of receptor-receptor interactions was the origin of the concept of meta-modulation provided by Katz and Edwards in 1999, which stood for the fine-tuning or modulation of nerve cell transmission. In 2000-2010, Ribeiro and Sebastiao, based on a series of papers, provided strong support for their view that adenosine can meta-modulate (fine-tune) synaptic transmission through adenosine receptors. However, another term should also be considered: protein modulation, which is the key feature of allosteric receptor-receptor interactions leading to learning and consolidation by novel adapter proteins to memory. Finally, it must be underlined that allosteric receptor-receptor interactions and their involvement both in brain disease and its treatment are of high interest. Their pathophysiological relevance has been obtained, especially for major depressive disorder, cocaine use disorder, and Parkinson's disease.

Defining the mechanism of galectin-3–mediated TGF-β1 activation and its role in lung fibrosis

Integrin-mediated activation of the pro-fibrotic mediator transforming growth factor-β1 (TGF-β1), plays a critical role in idiopathic pulmonary fibrosis (IPF) pathogenesis. Galectin-3 is believed to contribute to the pathological wound healing seen in IPF, although its mechanism of action is not precisely defined. We hypothesised that galectin-3 potentiates TGF-β1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-β1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L-α-lysophosphatidic acid sodium salt (LPA)-induced integrin-mediated TGF-β1 activation. Surface plasmon resonance (SPR) analysis confirmed that galectin-3 binds to αv integrins, αvβ1, αvβ5 and αvβ6 and to the TGFβRII subunit in a glycosylation-dependent manner. This binding is heterogeneous and not a 1:1 binding stoichiometry. Binding interactions were blocked by small molecule inhibitors of galectin-3 which target the carbohydrate recognition domain. Galectin-3 binding to β1 integrin was validated in vitro by co-immunoprecipitation in HLFs. Proximity ligation assays indicated galectin-3 and β1 integrin colocalize closely (≤40 nm) on the cell surface, that colocalization is increased by TGF-β1 treatment and blocked by galectin-3 inhibitors. In the absence of TGF-β1 stimulation, colocalization was detectable only in HLFs from IPF patients suggesting the proteins are inherently more closely associated in the disease state. Galectin-3 inhibitor treatment of precision cut lung slices from IPF patients reduced Col1a1, TIMP1 and HA secretion to a similar degree as TGF-β type I receptor inhibitor. These data suggest galectin-3 promotes TGF-β1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-β1 signaling cascade.

Phased nanopore assembly with Shasta and modular graph phasing with GFAse.

Reference-free genome phasing is vital for understanding allele inheritance and the impact of single-molecule DNA variation on phenotypes. To achieve thorough phasing across homozygous or repetitive regions of the genome, long-read sequencing technologies are often used to perform phased de novo assembly. As a step toward reducing the cost and complexity of this type of analysis, we describe new methods for accurately phasing Oxford Nanopore Technologies (ONT) sequence data with the Shasta genome assembler and a modular tool for extending phasing to the chromosome scale called GFAse. We test using new variants of ONT PromethION sequencing, including those using proximity ligation, and show that newer, higher accuracy ONT reads substantially improve assembly quality.