European Journal of Cell Biology

Intervertebral disc degeneration is associated with loss of nucleus pulposus (NP) cell phenotype and extracellular matrix, both processes linked to changes in cytoskeletal contractility and cell shape. Here, we tested whether microenvironment-specific modulation of RhoA signaling can restore NP-like morphology and gene expression in NP cells cultured in 2D and in 3D alginate. In 2D monolayer culture, where cells are spread and mechanically activated, pharmacologic inhibition of RhoA with CT04 reduced RhoA activity, decreased actomyosin contractility gene expression, and shifted morphology toward a smaller, more circular phenotype. Bulk RNA sequencing showed that CT04 treatment increased expression of NP phenotypic and matrix-related genes including ACAN, GDF5, CHST3, and MUSTN1 while decreasing expression of catabolic and fibroblast-associated genes including ADAMTS1/9 and COL1, consistent with enrichment of extracellular matrix pathways. In contrast, RhoA activation with CN03 in 2D culture increased actin and phosphorylated myosin light chain intensity but produced limited phenotypic improvement. In 3D alginate, which minimizes integrin-mediated adhesion, baseline actomyosin markers were reduced relative to 2D culture. In alginate, RhoA activation with CN03 increased the amount of actin, phosphorylated myosin light chain, and actomyosin gene expression, yet also promoted a more compact, circular morphology and increased NP markers, including ACAN and KRT19 with repeated dosing. Across culture conditions, increased cell roundness was consistently associated with increased ACAN expression, indicating strong coupling between cytoskeletal state, morphology, and NP matrix programs. Together, these findings demonstrate that RhoA pathway perturbation can promote NP phenotypic gene expression in both 2D and 3D culture, but the direction of optimal modulation depends on the microenvironment, supporting RhoA signaling as a context-dependent therapeutic target for disc regeneration.

Pathological cardiac remodeling is driven by the proliferation and differentiation of resident fibroblasts into active myofibroblasts and results in excessive extracellular matrix (ECM) deposition and tissue stiffening. Expression of the matricellular protein WISP1 has previously been shown to be increased with cardiac fibrosis and promote myofibroblast activity, but the mechanisms by which this occurs remain unknown. Primary cardiac fibroblasts were isolated from adult mouse hearts and treated with recombinant WISP1 or TGF{beta}1 both alone and in combination to determine the functional role of the matricellular protein WISP1 in driving cardiac myofibroblast activity. WISP1 significantly increased alpha-smooth muscle actin and collagen type I expression, total collagen secretion, collagen gel contractility, and wound healing equally in fibroblasts from both male and female mice. However, WISP1 alone failed to induce expression of periostin, a hallmark myofibroblast marker, suggesting the resulting WISP1-dependent cell phenotype is unique and/or acting through non-canonical pathways. Indeed, inhibition of P38 MAPK completely ablated the WISP1-dependent increase in SMA and collagen expression, while having little to no impact on TGF{beta}1-dependent expression of myofibroblast marker genes. We next employed a multi-omics approach to define the functional impact of WISP1 on fibroblast cell-state within the transcriptome, cytosolic, and secreted ECM proteome. RNA-seq results show that WISP1 broadly promotes the expression of proliferative and immune modulatory genes at the transcriptomic level, while having very little impact on traditional myofibroblast and ECM modifying gene expression programs. At the proteome level, WISP1 was again a much weaker mediator of traditional myofibroblast and ECM proteins. However, in agreement with RNA-seq data, we observed a strong WISP1-dependent enrichment for proliferation-associated proteins in the cytosolic proteome and inflammation-associated proteins in the ECM proteome. Interestingly, WISP1 also showed a context-dependent response with TGF{beta}1, suggesting a more complex and yet to be elucidated signaling interaction between these independent mediators of myofibroblast activity. In conclusion, our data suggests that WISP1 promotes a unique proliferative and immune-modulatory myofibroblast phenotype. HighlightsO_LIWISP1 is sufficient to drive myofibroblast SMA and collagen expression and ECM deposition C_LIO_LIWISP1 promotes canonical myofibroblast contractility and wound healing activity C_LIO_LIWISP1 mediates myofibroblast activity via a non-canonical, P38 MAPK-dependent signaling pathway C_LIO_LIMulti-omics analysis of WISP1-dependent RNA and protein expression show that WISP promotes a proliferative and immune modulatory myofibroblast phenotype C_LI

The extracellular matrix (ECM) is a meshwork of proteins that orchestrates a broad range of cellular phenotypes, including proliferation, adhesion, migration, and differentiation. SNED1 is a newly characterized ECM glycoprotein that promotes cell adhesion and is essential for embryonic development. Its upregulation is also associated with breast cancer metastasis and poor prognosis for breast cancer patients. We recently showed that SNED1 assembles into fibrillar structures, but the mechanisms guiding its incorporation into the ECM scaffold remain unknown. Combining biochemical assays and confocal immunofluorescence imaging, we found that SNED1 assembly in the ECM occurs early in the process of ECM building and is concomitant and overlaps with the deposition of fibronectin and collagen I, two major ECM proteins. By knocking down fibronectin or destabilizing collagen I fibers, we further demonstrate that SNED1 requires the presence of these proteins for its assembly. Last, using biolayer interferometry, we identify collagen I as the first direct binding partner of SNED1. Altogether, our results lay the foundation for future studies aimed at determining the mechanisms by which SNED1 fibers contribute to SNED1 pathophysiological functions. SUMMARY STATEMENTThe novel protein SNED1 requires the presence of fibronectin and collagen I to assemble into fibrillar structures in the extracellular matrix scaffold.

Cartilage is characterized by a highly specialized extracellular matrix (ECM) secreted by chondrocytes and limited self-regenerative capacity. In vivo investigations of chondrogenesis are limited by difficult and traumatic access, especially in humans. While it is known for decades that disturbances of chondrocyte differentiation and changed cartilage ECM composition cause severe skeletal phenotypes in vertebrates, a detailed molecular understanding of chondrogenesis and cartilage ECM formation is still missing, especially in the context of human genetic skeletal diseases. ATDC5 cells, derived from AT805 mouse teratocarcinoma cells, have been used in the past to model chondrogenic differentiation, however, most studies have investigated few major cellular differentiation markers only so that the composition of the secreted ECM as well as effects on the ATDC5 transcriptome upon differentiation are still unclear. Here, we performed time-resolved transcriptomic and ECM proteomic analyses of differentiating ATDC5 cells. Both datasets confirmed the formation of a cartilage-like matrix with increasing expression of key chondrocyte genes over the course of differentiation. ECM proteomics further revealed a number of ECM components not previously reported in ATDC5 cells or the secreted ECM, encompassing collagens, proteoglycans, glycoproteins and other secreted factors. Overall, our findings provide a more detailed molecular characterization of ATDC5 chondrogenesis and highlight the potential of this model system for ECM-focused studies.

Mechanosensing involves proteins detecting mechanical changes in the cytoskeleton or at cell adhesion sites. These interactions initiate signaling cascades that produce biochemical effects such as post-translational modifications or cytoskeletal rearrangements. Filamin is a ubiquitous mechanosensing protein that binds actin filaments and senses pulling forces within the cytoskeleton. Drosophila Filamin (Cheerio) is structurally similar to mammalian Filamin, with roles in egg chamber development, embryo cellularization, and integrity of muscle attachment sites and Z discs in Drosophila indirect flight muscles (IFMs). Here we report a potential novel binding partner of Drosophila Filamins: the death-associated protein kinase Drak that functions as a myosin light chain kinase. We found that Drak biochemically bound to an open mutant of Filamin that resembles the mechanically activated form partially bound to wild type Filamin and did not bind to closed mutant of Filamin. The interaction site was mapped to the intrinsically unfolded C-terminal region of Drak. To study the functional role of Drak-Filamin interaction, we studied two developmental events where Drak has been earlier shown to be expressed and where Filamin also functions: early embryonic cellularization and indirect flight muscle development at pupal stages. We found partial colocalization between Drak-GFP and Filamin-mCherry during the initiation of cellularization furrow, and at the time of myotube attachment site maturation in tendon cells. However, functionally we could not show direct correlation between Filamin and Drak. Our studies reveal interesting new expression patterns of Drak during Drosophila development and provide detailed information about Filamin localization during IFM development.

Galectin-3 (Gal3) is one of the most pro-inflammatory proteins and a biomarker of inflammatory diseases and cancer. Previous studies showed that Gal3 binds to v and {beta}1 integrins but it is unclear how Gal3 binds to integrins. Here, we show that Gal3 bound to soluble v{beta}3 and IIb{beta}3 integrins in 1 mM Mn2+ in cell-free conditions in a glycan-independent manner. Docking simulation predicts that Gal3 binds to the classical RGD-binding site (site 1) of v{beta}3, but the predicted Gal3-binding site does not include galactose-binding site. RGDfV or eptifibatide inhibited Gal3 binding to v{beta}3 and IIb{beta}3, respectively, but lactose, pan-galectin inhibitor, did not inhibit Gal3 binding to integrins. Point mutations of the predicted site 1 binding interface of Gal3 effectively inhibited Gal3 binding to site 1. Site 2 is involved in pro-inflammatory signaling (e.g., TNF and IL-6 secretion) and we previously showed that pro-inflammatory cytokines (e.g., CCL5 and TNF) bind to site 2 and allosteric integrin activation. Docking simulation predicts that Gal3 binds to site 2 of v{beta}3 and 5{beta}1. We found that Gal3 induced allosteric activation of soluble integrins v{beta}3, IIb{beta}3, and 5{beta}1 in 1 mM Ca2+ in cell-free conditions. Point mutations in the predicted site 2-binding interface inhibited Gal3-induced integrin activation, suggesting that Gal3 binding to site 2 is required for Gal3-induced integrin activation. Known anti-inflammatory agents, Ivermectin, NRG1, and FGF1 inhibited integrin activation induced by Gal3 in v{beta}3 and IIb{beta}3. These findings suggest that Gal3 binding to site 2 may be a potential mechanism of pro-inflammatory and pro-thrombotic action of Gal3.

Ex vivo models bridge in vitro and in vivo systems by preserving native extracellular matrix architecture and multicellular interactions. In articular joint research, osteochondral-synovial co-cultures are particularly valuable for studying bone-cartilage crosstalk and synovial inflammatory regulation. However, a lack of standardized culture conditions regarding glucose and oxygen, two key regulators of cellular metabolism, limits reproducibility and translational relevance. This study aims to define how glucose and oxygen conditions influence joint tissues maintenance in an ex vivo model. Bovine osteochondral explants and synovium are harvested from the stifle joint and co-cultured using either high glucose DMEM (HG, 4.5 g/L) or low glucose DMEM (LG, 1 g/L) under hyperoxic (21% O2) or physioxic (5% O2) conditions. Cell viability, gene expression, and metabolomic profiles are evaluated across tissues. LG conditions increase cell death in the deep zone of cartilage and in subchondral bone. Gene expression and metabolomic analyses reveal tissue-specific effects of glucose and oxygen. In cartilage and bone, glucose-dependent gene regulation and metabolic changes occur under hyperoxia but are largely absent under physioxia, indicating buffering of glucose responses. Gene-specific sensitivity to glucose and oxygen is observed in bone and synovium; however, glucose-induced metabolic responses persist under physioxia only in synovium. Overall, these findings identify oxygen and glucose as critical modulators of joint tissue physiology and support the use of HG, physioxic culture conditions to improve cell viability and stabilize molecular outcomes in ex vivo joint models. This optimized ex vivo model provides platforms for investigating mechanisms relevant to joint-related diseases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/704322v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1393324org.highwire.dtl.DTLVardef@4c9393org.highwire.dtl.DTLVardef@16cc00eorg.highwire.dtl.DTLVardef@b4d9ea_HPS_FORMAT_FIGEXP M_FIG C_FIG This study evaluates the effects of glucose concentration and oxygen tension in an ex vivo joint co-culture system to define optimal culture conditions. High glucose medium and physioxic conditions support tissue viability, preserve homeostasis, and enhance the physiological relevance of the ex vivo model.

Cellular transport processes along microtubules are often facilitated by multi-motor complexes, which are connected by adapter proteins and cargoes. The nuclear pore protein Nup358, for example, interacts with the dynein adapter Bicaudal D2 (BicD2), which in turn recruits minus-end directed dynein motors and plus-end directed kinesin-1 motors for a nuclear positioning pathway that is essential for brain development. How motor recruitment is regulated by interactions of BicD2 with Nup358 is not well understood. Here, we characterize the structure of a minimal complex of kinesin-1 light chain 2 (KLC2), Nup358 and BicD2 by cryo-electron microscopy and small angle X-ray scattering. KLC2/Nup358 assumes a rod-like structure that increases in thickness, when BicD2 is bound. The addition of BicD2 also shifts the KLC2/Nup358/BicD2 complex towards a 2:2:2 stoichiometry, promoting dimerization at lower protein concentrations than without BicD2. Similarly, the presence of the Nup358/KLC2 interaction results in a shift towards a 2:2:2 stoichiometry. Based on these results, we hypothesize that KLC2 and BicD2 are recruited to Nup358 in a cooperative manner, and cooperativity may be promoted by modulation of the oligomeric state.

Variants in ACTB gene encoding for cytoplasmic {beta}-actin result in a group of rare disorders called non-muscle actinopathies (NMA). We investigated the cellular effects of a missense variant, G302A, and a four-amino-acid deletion, S338-I341, associated with the subgroup of NMA - ACTB pLoF (predicted loss-of-function) disorder in patient-derived fibroblast cells. We found that neither of the mutations affected the organization of actin or the width of the actin-filament bundles, while the mutation G302A reduced the stiffness of the cells as measured by using atomic force microscopy. The latter effect might be associated with the misorganization of tubulin and with the increased size and number of focal adhesions. When we challenged the cells by monolayer stretching and followed the mechanically-induced reorganization of the actin cytoskeleton, we found that G302A mutant cells showed more dense actin filament bundles within the cells compared to wild type cells. At the same time, the extent of cofilin reorganization from the cell periphery was increased upon stretch, and this correlated with an increased cofilin phosphorylation. In the case of the deletion, while the extent of cofilin phosphorylation increased, the extent of reorganization was unaltered; rather, the phosphorylation of myosin light chain, important in counteracting external force, was drastically reduced. We could partially rescue this fascinating effect by overexpressing the active form of the formin mDia. Our findings open the possibility to validate the cellular phenotype in the most affected patients cells, in neurons.

Envelope-Limited Chromatin Sheets (ELCS) can be induced in human promyelocytic HL-60/S4 cells by treatment with retinoic acid (RA). After 4 days, the differentiated granulocytes exhibit multilobed nuclei with outgrowths of the nuclear envelope (NE) and associated heterochromatin extending into the surrounding cytoplasm (ELCS). These fascinating structures reveal a periodic meshwork of 30 nm chromatin fibers, when viewed by Cryo-electron microscopy. Genetic and biochemical evidence indicates that RA increases the synthesis of Lamin B Receptor (LBR), which is a key enzyme for Cholesterol biosynthesis and is an essential bridge between the NE and peripheral heterochromatin. This article is in part a review of our microscopic data on the structure of ELCS, and in part a description of related transcription changes that result in the formation of ELCS. In addition, this article contains a structural and biochemical comparison of RA-induced granulocytes with phorbol ester (TPA) induced HL-60/S4 macrophages, which lack nuclear lobulation, do not form ELCS, and exhibit a reduction in LBR and Cholesterol biosynthesis. From our perspective, ELCS can be viewed as "fabric" outgrowths of the nuclear envelope, frequently connecting nuclear lobes and capable of sustaining the twisting and squeezing distortions imposed upon nuclear shape, as the granulocytes traverse narrow tissue channels.

Brain metastases are a common and often fatal complication of certain cancer types, such as triple-negative breast cancer. However, the molecular pathways driving brain metastasis formation, including the migration of cancer cells from the bloodstream to the brain parenchyma across the blood-brain barrier, are not yet fully defined. Therefore, using highly relevant mouse and human model systems, the mechanisms by which triple-negative breast cancer cells and their released extracellular vesicles modulate the blood-brain barrier-forming endothelium to increase its permissiveness to tumour cell entry into the brain are investigated. It is observed that extracellular vesicles derived from tumour cells are taken up by cerebral endothelial cells, where they induce miR-146a-5p- and TGF-{beta}1-mediated downregulation of PAQR5/mPR{gamma}, a membrane progesterone receptor. This, in turn, leads to disruption of interendothelial tight junctions, particularly through repression of claudin-5 expression, a critical protein for maintaining barrier function. Altogether this identifies a novel mechanism by which triple-negative breast cancer-derived extracellular vesicles compromise blood-brain barrier integrity, thereby facilitating transendothelial migration of cancer cells and promoting brain metastasis development. Moreover, this study is the first to highlight the role of membrane progesterone receptors in regulating the blood-brain barrier. Table of contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/701753v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@15252aeorg.highwire.dtl.DTLVardef@1b23beforg.highwire.dtl.DTLVardef@7cd517org.highwire.dtl.DTLVardef@189db4e_HPS_FORMAT_FIGEXP M_FIG Extracellular vesicles from triple-negative breast cancer cells induce miR-146a-5p- and TGF-1-mediated downregulation of PAQR5/mPR{gamma}, a membrane progesterone receptor, in blood-brain barrier-forming endothelial cells. This results in disruption of interendothelial tight junctions, thereby promoting enhanced migration of cancer cells into the brain. This mechanism highlights the role of membrane progesterone receptors in regulating the blood-brain barrier. C_FIG

Nicotinic acetylcholine receptors (nAChRs) belong to the pentameric ligand-gated ion channel superfamily (pLGICs). Among them, the neuronal homomeric 7 nAChR is highly permeable to calcium and plays critical roles in synaptic transmission, cell signaling, and inflammation modulation. The biogenesis of 7 nAChRs is enhanced by the chaperone proteins RIC-3 and NACHO. Previously, we reported a motif in the 5-HT3A receptor, another pLGIC, involved in RIC-3 modulation. Residues in this motif are conserved and also found within the L1-MX segment of the 7 nACh subunit. We therefore explored the regulatory roles of these conserved residues in the biogenesis of 7 nAChRs using multiple approaches, including heterologous expression in Xenopus laevis oocytes, mutagenesis, pull-down assays, cell-surface labeling, and two-electrode voltage-clamp (TEVC) recordings. We find that synthetic 7 L1-MX peptide interacts with both RIC-3 and NACHO. In particular, conserved residues W330, R332, and L336 in the L1-MX positively regulates the assembly of 7 oligomers and the biogenesis of 7nAChR. In presence of residues W330, R332, and L336, NACHO promotes an assembly of an 7 pentamer which is resistant to strong denaturing conditions. NACHO-promoted 7 pentamer is also resistant to Endo H enzyme. Sensitivity of the pentamer to moderate temperatures (37 {degrees}C, 45 {degrees}C, and 50 {degrees}C) suggests that NACHO stabilizes the pentamer via non-covalent interactions. In contrast, Ala replacements at these residues disrupt the biogenesis and abolish 7 current. NACHO and RIC-3 co-expression yields partial rescue of functional expression for some Ala replacement constructs. SUMMARYThis work identifies regulatory roles of conserved residues W330, R332, and L336 in the biogenesis of 7 nAChR. This discovery positions MX subdomain as a promising target for future drug development that can minimize adverse effects.

The intracellular transport system is pivotal for cellular function and integrity, facilitated by cytoskeletal motor proteins such as dynein, which traverse along microtubules (MTs). The heterogeneity of the tubulin isotypes composing MTs introduces functional diversity, potentially affecting cytoskeletal motor proteins interactions with the MT. This in silico study investigated the influence of amino acid sequence variations in the C-terminal tails (CTTs) of six different Homo sapiens tubulin isotypes, TUBB2A, TUBB2B, TUBB2C, TUBB3, TUBB4A, and TUBB5, highly expressed in human brain tumors, and assessed the isotypes effect on the binding of motor protein dynein to MT. Among these isotypes, TUBB2A, TUBB2B, and TUBB2C were found to affect conformational motions of the dyneins microtubule-binding domain (MTBD) and stalk domain. The investigation highlighted the novel role of isotype-specific variations in lateral interactions between tubulin protofilaments (PFs) in determining the proximity of the {beta}-CTT of the adjacent PF to the MTBD, potentially affecting dyneins motility and suggesting how changes in isotype expression directly influence dyneins velocity and processivity and contribute to transport defects associated with neurological disorders and cancers. Isolating specific tubulin isotypes experimentally is challenging due to their high sequence similarity and complex interactions with other microtubule-associated proteins. This makes it challenging to distinguish between different tubulin isotypes and their effects, particularly in tissues where multiple isotypes are co-expressed. Additionally, these isotypes are heavily modified in vivo by post-translational modifications, which further complicate the isolation of a single, unmodified tubulin isotype. As a result, computational approaches have been necessary in this study for exploring these effects in a controlled, isotype-specific manner.

The extracellular matrix (ECM) is a complex scaffold of proteins that supports multicellular structures. Interactions between cells and the ECM via receptors, like integrins, govern cellular phenotypes (e.g., proliferation, adhesion), but also contribute to ECM assembly. Understanding how ECM-receptor interactions regulate matrix assembly is critical to uncover how alterations of the ECM cause or accompany congenital diseases, cancer, or fibrosis. SNED1 is a novel ECM protein with roles in development and metastasis. However, the mechanisms governing its assembly and signaling functions remain largely unknown. SNED1 contains two integrin-binding motifs, RGD and LDV, and we recently showed that its interaction with RGD-integrins mediates cell adhesion. Here, we investigated the role of SNED1/integrin interactions in SNED1 ECM assembly. While SNED1/integrin interactions were not necessary for its initial incorporation in the ECM, interaction with LDV-, but not RGD-, integrins, was required for ECM build-up and the patterning of SNED1 and the fibrillar proteins fibronectin and collagen I. Moreover, SNED1/LDV-integrin interaction promoted ECM alignment, cell alignment, and cell proliferation, processes essential to SNED1-driven neural crest cell migration during craniofacial development and breast cancer invasion. SUMMARY STATEMENTInteraction of SNED1 with LDV-binding integrins, but not RGD-binding integrins, mediates ECM remodeling and controls cytoskeletal rearrangement and cell proliferation.

Motile cells can sense and exert forces on the extracellular environment through dynamic actin networks. Increased stress against the polymerizing barbed ends of branched actin networks has been shown to lead to an increase in the density of these networks through a force feedback mechanism, though this phenomenon has not been explored through the examination of real-time responses of endogenous actin networks in cells. Here, we utilize mouse embryonic fibroblast CRISPR knock-in lines with labeled ARP2/3 complex to identify cellular and extracellular conditions that regulate branched actin density and enrichment at the leading edge of lamellipodial protrusions. A common theme shared among all branched actin density-increasing conditions is higher levels of interface stress between the plasma membrane and the barbed ends of the lamellipodial actin network. Among these conditions, we find that ARP2/3 is specifically required for robust spreading and protrusion in response to increased extracellular viscosity. Interestingly, time-lapse traction force microscopy of ARP2/3-dependent viscosity responses show significantly reduced changes in strain energy applied to the substrate when compared to spreading and motility through cell-matrix adhesion. In addition, we find that increased extracellular viscosity can bypass the need for extracellular matrix proteins to support lamellipodial protrusion driven by optogenetic Rac activation. Our studies provide strong support for in vitro models of branched actin force feedback responses and further characterize an essential role for branched actin in mediating dramatic cell shape changes in response to increased extracellular viscosity.

BackgroundIn vertebrates, skeletal muscle grows postnatally through different strategies. While mammals predominantly rely on fiber hypertrophy after birth, many teleost fish retain the unique ability to generate new fibers via hyperplasia well into juvenile stages. The molecular mechanisms governing the transition between hyperplastic-hypertrophic and hypertrophic growth modes in fish muscle remain poorly understood. ResultsWe generated a single-cell transcriptomic atlas of muscle-derived cells from juvenile Oncorhynchus mykiss (rainbow trout) at five growth stages. Fifteen tissue resident cell populations were identified, including eight myogenic subpopulations spanning from quiescent stem cells to terminally differentiating myocytes. Two distinct transcriptional trajectories were uncovered thanks to RNA velocity analysis: one present only during hyperplastic growth and another maintained throughout growth, indicating specialization of satellite cells toward hyperplasia or hypertrophy. Comparative analyses with human single-cell atlases indicate that subpopulations specifically related to hyperplasia and hypertrophy are conserved, depending on stage (fetal or adult). Strikingly, we identified a population of pax7+/pdgfr+ cells, indicating plasticity toward fibroblastic lineage and associating these cells with hypertrophic growth. Furthermore, both intrinsic changes in muscle stem cells and extrinsic remodeling of the extracellular matrix accompanied the decline of hyperplasia, highlighting dynamic crosstalk between myogenic and mesenchymal compartments. ConclusionsOur findings reveal the existence of two transcriptionally distinct muscle stem cell fates that underlie hyperplastic versus hypertrophic growth in fish. The identification of a tissue-resident pax7+/pdgfr+ subpopulation provides new insights into muscle stem cell plasticity and niche remodeling. This work establishes a comparative framework to explore the regulation of postnatal muscle growth across vertebrates.

BackgroundPeripheral nerve trauma denervates skeletal muscle resulting in paralysis and atrophy that is reversible if timely reinnervation occurs, due to its regenerative capacity. If reinnervation is delayed muscles regenerative ability is exhausted and resident fibroadipogenic progenitors (FAPs) differentiate into adipocytes and fibroblasts that replace muscle with non-contractile fibrotic tissue and fat, resulting in physical disability. Prostaglandin E2 (PGE2) inhibits adipogenesis and fibrosis in other tissues. We determined whether PGE2 could inhibit fibro-fatty degradation of long-term denervated muscle. MethodsWe utilized the rat tibial nerve transection model, denervating the gastrocnemius and selected a 5 week post-denervation time point to represent short-term muscle denervation injury (reversible with reinnervation), and 12 weeks to represent sustained, irreversible injury. Gastrocnemius FAPs were isolated via FACS and grown in culture to assess endogenous PGE2 production and the proliferative and differentiation response to exogenous PGE2. We evaluated transcript and protein expression of PGE2 synthesizing enzyme PTGS2, PGE2 degrading enzyme 15-PGDH and markers of proliferation, adipogenesis and fibrogenesis using RT-qPCR, immunofluorescence and SDS-PAGE/Western blotting. Paracrine impact of FAPs produced PGE2 was assessed by treating C2C12 myoblasts with FAPs conditioned media. ResultsTranscript expression of PTGS2 was increased and 15-PGDH decreased (4.37{+/-}2.63 and -3.06{+/-}0.85 fold change respectively, p<0.05) in 5 week, but not 12 week denervated gastrocnemius, consistent with increased PGE2 production in 5 week denervated muscle. Similarly, PTGS2 transcript levels were significantly increased (2.58{+/-}0.33 fold change, p<0.05) and 15-PGDH decreased (-5.24{+/-}3.19 fold change, p<0.05) in FAPs isolated from 5 week, but not 12 week denervated muscle, demonstrating that FAPs are a source of PGE2 in short-term denervated muscle. 16,16-dimethyl PGE2 did not impact naive FAPs in vitro proliferation, but significantly inhibited their differentiation as demonstrated by 88.9%, 82.3% and 94.2% decreases in FAPs expression of adipogenic marker perilipin-1, fibrogenic marker -smooth muscle actin (-SMA) and lipid content respectively, mediated via PGE2 binding to the FAPs EP4 receptor. FAPs isolated from 12 week denervated muscle demonstrated increased adipogenesis and fibrogenesis vs. naive FAPs (perilipin-1 and -SMA 7.93{+/-}2.96 and 2.00{+/-}0.33 fold increase respectively, p<0.05) and remained fully susceptible to PGE2 inhibition of fibro-adipogenic differentiation. Conditioned media from FAPs derived from 5 week, but not 12 week, denervated gastrocnemius stimulated C2C12 myoblast proliferation which was prevented by EP4 blockade. ConclusionsPGE2 is identified as a novel negative regulator of FAPs differentiation in traumatically denervated muscle, suggesting the therapeutic potential of PGE2 to prevent fibro-fatty degradation of long-term denervated muscle awaiting reinnervation.

The blood-brain barrier (BBB) protects the brain from circulating metabolites and plays central roles in neurological diseases. Endothelial cells (ECs) of the BBB are enwrapped by mural cells including pericytes and vascular smooth muscle cells (vSMCs) that regulate angiogenesis, vessel stability and barrier function. To explore mural cell control of the BBB, we investigated neurovascular phenotypes in zebrafish pdgfrb mutants that lack brain pericytes and vSMCs. As expected, mutants showed an altered cerebrovascular network with mispatterned capillaries. Unexpectedly, mutants displayed no BBB leakage at larval stages of development. This demonstrates that pericytes and vSMCs do not control BBB function in developing zebrafish. Instead, we observed juvenile and adult BBB disruption occurring at "hotspot" focal hemorrhages at large vessel aneurysms. ECs at leakage hotspots showed induction of caveolae on abluminal surfaces and structural defects including basement membrane thickening and disruption. Our work suggests that capillary pericytes regulate cerebrovascular patterning in development and vSMCs of major arteries protect from hemorrhage and BBB breakdown in older zebrafish. The fact that young zebrafish have a functional BBB in the absence of mural cells calls for renewed interrogation of mural cell control of the BBB throughout vertebrate evolution.

It has been recognized a long time ago that the hedgehog (Hh) and Wnt signaling pathways have numerous similarities that suggest their common evolutionary origin. Although the Hh and Wnt proteins are unrelated they are similar in as much as they carry lipid modifications that are critical for their interaction with their receptors. In our earlier work we have shown that Wnt inhibitory factor 1 (WIF1), originally identified as a Wnt antagonist also binds to and inhibits the signaling activity of sonic hedgehog (Shh), raising the possibility that the lipid moieties of these unrelated morphogens play a dominant role in their interaction with WIF1. In the present work we have compared the interactions of human WIF1 protein with lipidated and non-lipidated forms of human sonic hedgehog (Shh) using Surface Plasmon Resonance spectroscopy and reporter assays monitoring the signaling activity of human Shh. Our studies have shown that human WIF1 protein has significantly higher affinity for lipidated than non-lipidated Shh, indicating that lipid modifications of Hhs are important for interactions with WIF1.

BackgroundDexamethasone (DEX) is used in vitro to promote osteogenic differentiation of human bone marrow mesenchymal stromal cells (hBMSCs). In clinical use, however, glucocorticoids induce osteoblast and osteocyte apoptosis while increasing osteoclast survival, leading overall to osteoporosis and high fracture risk. The overall impact of DEX on the differentiation of human progenitor cells remains contradictory and not fully understood, highlighting the need for further investigation using sequencing approaches as in vitro results will naturally influence further translational research. MethodshBMSCs were induced to osteogenic differentiation for 7 days using different concentrations of either DEX or the nonsteroidal glucocorticoid receptor agonist (+)-ZK216348. cDNA library preparation and RNA sequencing (RNAseq) were performed using Oxford Nanopore Technologies. Differentially expressed genes and pathways associated to the transactivation or transrepression activity of DEX were identified. Sequencing results were validated by qPCR, protein analysis, and with a functional assay on peripheral blood mononuclear cells to determine the overall effect of the BMSC supernatant. ResultsHierarchical clustering of RNAseq data identified eight subclusters with shared regulatory patterns. Enrichment analysis revealed that both upregulated and downregulated genes are involved in ossification and extracellular matrix organization pathways. Several pro- and anti-inflammatory genes were differentially regulated. qPCR analysis validated the upregulation of CXCL1, CXCL8, IL18, and COL8A1, while MMP1 and CXCL12 expression decreased in response to DEX. Comparing DEX results with those obtained using (+)-ZK216348 helped distinguish the potential mechanisms regulating the expression of specific genes. Notably, CXCL8 upregulation occurred through transactivation, whereas COL8A1 upregulation is downstream of a transrepressed gene. Further in vitro experiments confirmed that DEX significantly increased CXCL8 expression and IL-8 secretion. However, hPBMC responses indicated no significant pro- or anti-inflammatory effects from hBMSC conditioned medium. ConclusionsIn conclusion, the effects of DEX on the transcriptome of hBMSCs in a pro-osteogenic environment do not fully replicate the acquisition of an osteogenic phenotype. Several genes associated with ossification, extracellular matrix organization, and inflammation were dysregulated. The unique expression patterns of pro-inflammatory cytokines and collagen types warrant further investigation to elucidate their roles in osteogenic differentiation and bone homeostasis.