The FASEB Journal

Skeletal muscle is highly plastic and capable of remodelling its contractile and metabolic properties depending on physical demands. Such remodelling requires modification of chromatin structure to support transcriptional activation and suppression of gene programs. Chromatin dynamics depend, in part, on the acetylation and methylation of histone 3 lysine 27 (H3K27), which is controlled by several H3K27-specific acetyltransferases, deacetylases, methyltransferases and demethylases. Several histone post-translational modifications in muscle have been shown to be modulated by exercise. Here, we sought to examine whether major H3K27 regulators are altered by endurance training. Male and female C57BL/6J mice were provided with voluntary running wheels for 6 weeks and compared to sex-matched sedentary controls with locked running wheels. Gene expression of various slow/oxidative and fast/glycolytic skeletal muscles was then measured. We found voluntary running induced modest changes in H3K27 acetyltransferases, along with several components of the polycomb-repressive complex 2 in a muscle- and sex-specific manner. Our findings indicate that the capacity for both acetylation and methylation of H3K27 is modulated by chronic endurance exercise, suggesting that chromatin dynamics are a mechanistic component of exercise-induced muscle remodelling.

Acute tendon injuries are characterized by excessive matrix deposition that impedes regeneration and disrupts functional improvements. Inflammation is postulated to drive pathologic scar tissue formation, with nuclear factor kappa B (NF-{kappa}B) signaling emerging as a candidate pathway in this process. However, characterization of the spatial and temporal activation of canonical NF-{kappa}B signaling during tendon healing in vivo, including identification of the cell populations activating NF-{kappa}B, is currently unexplored. Therefore, we aimed to determine which cell populations activate canonical NF-{kappa}B signaling following flexor tendon repair with the goal of delineating cell-specific functions of NF-{kappa}B signaling during scar mediated tendon healing. Immunofluorescence revealed that both tendon cells and myofibroblasts exhibit prolonged activation of canonical NF-{kappa}B signaling into the remodeling phase of healing. Using cre-mediated knockout of the canonical NF-{kappa}B kinase (IKK{beta}), we discovered that suppression of canonical NF-{kappa}B signaling in Scleraxis-lineage cells increased myofibroblast content and scar tissue formation. Interestingly, Scleraxis-lineage specific knockout of IKK{beta} increased the incidence of apoptosis, suggesting that canonical NF-{kappa}B signaling may be mediating cell survival during tendon healing. These findings suggest indispensable roles for canonical NF-{kappa}B signaling during flexor tendon healing. One Sentence SummaryScleraxis-lineage specific knockdown of persistent canonical IKK{beta}/NF-{kappa}B drives scar formation and apoptotic signaling during flexor tendon healing.

Chronic kidney disease (CKD) is characterized by muscle atrophy, fatigue intolerance and other indicators of muscle dysfunction, collectively termed uremic myopathy, with devastating consequences in overall health status and mortality rates. Although many factors such as metabolic acidosis, substrate availability and neuropathy have been implicated, the mechanisms underlying uremic myopathy have not yet been fully understood. However, there is clear evidence that muscle specific factors such as fiber atrophy, fiber type alterations and mitochondrial abnormalities are presented in muscle biopsies of CKD patients and can negatively affect muscle contraction. Counteracting measures such as exercise and nutritional interventions have been shown to improve muscle performance, health indices and overall quality of life of CKD patients. However, little is known about their effects on factors affecting muscle contraction at the muscle biopsy level and therefore on the mechanisms underlying uremic myopathy. The current systematic review aims to summarize the effects of recent interventional studies on muscle contraction determinants based on muscle biopsies of human patients.

The actin cytoskeleton is a potent regulator of tenocyte homeostasis. However, the mechanisms by which actin regulates tendon homeostasis are not entirely known. This study examined the regulation of tenocyte molecule expression by actin polymerization via the globular (G-) actin-binding transcription factor, myocardin-related transcription factor-a (MRTF). We determined that decreasing the proportion of G-actin in tenocytes by treatment with TGF{beta}1 increases nuclear MRTF. These alterations in actin polymerization and MRTF localization coincided with favorable alterations to tenocyte gene expression. In contrast, latrunculin A increases the proportion of G-actin in tenocytes and reduces nuclear MRTF, causing cells to acquire a tendinosis-like phenotype. To parse out the effects of F-actin depolymerization from regulation by MRTF, we treated tenocytes with cytochalasin D. Similar to latrunculin A treatment, exposure of cells to cytochalasin D increases the proportion of G-actin in tenocytes. However, unlike latrunculin A treatment, cytochalasin D increases nuclear MRTF. Compared to latrunculin A treatment, cytochalasin D led to opposing effects on the expression of a subset of genes. The differential regulation of genes by latrunculin A and cytochalasin D suggests that actin signals through MRTF to regulate a specific subset of genes. By targeting the deactivation of MRTF through the inhibitor CCG1423, we verify that MRTF regulates Type I Collagen, Tenascin C, Scleraxis, and -smooth muscle actin in tenocytes. Actin polymerization status is a potent regulator of tenocyte homeostasis through the modulation of several downstream pathways, including MRTF. Understanding the regulation of tenocyte homeostasis by actin may lead to new therapeutic interventions against tendinopathies, such as tendinosis.

The actin cytoskeleton is a central mediator between mechanical force and cellular phenotype. In tendon, it is speculated that mechanical stress deprivation regulates gene expression by filamentous (F-) actin destabilization. However, the molecular mechanisms that stabilize tenocyte F-actin networks remain unclear. Tropomyosins (Tpms) are master regulators of F-actin networks. There are over 40 mammalian Tpm isoforms, with each isoform having the unique capability to stabilize F-actin sub-populations. Thus, the specific Tpm(s) expressed by a cell defines overall F-actin organization. Here, we investigated F-actin destabilization by stress deprivation of tendon and tested the hypothesis that stress fiber-associated Tpm(s) stabilize tenocyte F-actin to regulate cellular phenotype. Stress deprivation of mouse tail tendon fascicles downregulated tenocyte genes (collagen-I, tenascin-C, scleraxis, -smooth muscle actin) and upregulated matrix metalloproteinase-3. Concomitant with mRNA modulation were increases in DNAse-I/Phallodin (G/F-actin) staining, confirming F-actin destabilization by tendon stress deprivation. To investigate the molecular regulation of F-actin stabilization, we first identified the Tpms expressed by mouse tendons. Tendon cells from different origins (tail, Achilles, plantaris) express three isoforms in common: Tpm1.6, 3.1, and 4.2. We examined the function of Tpm3.1 since we previously determined that it stabilizes F-actin stress fibers in lens epithelial cells. Tpm3.1 associated with F-actin stress fibers in native and primary tendon cells. Inhibition of Tpm3.1 depolymerized F-actin, leading to decreases in tenogenic expression, increases in chondrogenic expression, and enhancement of protease expression. These expression changes by Tpm3.1 inhibition are consistent with tendinosis progression. A further understanding of F-actin stability in musculoskeletal cells could lead to new therapeutic interventions to prevent alterations in cellular phenotype during disease progression.

There is limited understanding of how mechanical signals regulate tendon development. The nucleus has emerged as a major regulator of cellular mechanosensation, via the linker of nucleoskeleton and cytoskeleton (LINC) protein complex. Specific roles of LINC in tenogenesis have not been explored. In this study, we investigate how LINC regulates tendon development by disabling LINC-mediated mechanosensing via dominant negative (dn) expression of the Klarsicht, ANC-1, and Syne Homology (KASH) domain, which is necessary for LINC to function. We hypothesized that LINC regulates mechanotransduction in developing tendon, and that disabling LINC would impact tendon mechanical properties and structure in a mouse model of dnKASH. We used Achilles (AT) and tail (TT) tendons as representative energy-storing and limb-positioning tendons, respectively. Mechanical testing at postnatal day 10 showed that disabling the LINC complex via dnKASH significantly impacted tendon mechanical properties and cross-sectional area, and that effects differed between ATs and TTs. Collagen crimp distance was also impacted in dnKASH tendons, and was significantly decreased in ATs, and increased in TTs. Overall, we show that disruption to the LINC complex specifically impacts tendon mechanics and collagen crimp structure, with unique responses between an energy-storing and limb-positioning tendon. This suggests that nuclear mechanotransduction through LINC plays a role in regulating tendon formation during neonatal development.

Skeletal muscle adaptation to exercise involves various phenotypic changes that enhance metabolic and contractile functions. One key regulator of these adaptive responses is the activation of AMPK, influenced by exercise intensity. However, the mechanistic understanding of AMPK activation during exercise remains incomplete. In this study, we utilized an in vitro model to investigate the effects of mechanical loading on AMPK activation and its interplay with the mTOR signaling pathway. Proteomic analysis of myoblasts subjected to static loading (SL) revealed distinct quantitative protein alterations associated with RNA metabolism, with 10% SL inducing the most pronounced response compared to lower intensity of 5% and 2% as well as control. Additionally, 10% SL suppressed RNA and protein synthesis, while activating AMPK and inhibiting the mTOR pathway. Our RNA sequencing analysis further corroborated these findings, revealing numerous differentially regulated genes and signaling pathways influenced by both AMPK and mTOR. Further examination showed that SL induced changes in mitochondrial biogenesis and the ADP/ATP ratio. These findings provide novel insights into the cellular responses to mechanical loading and shed light on the intricate AMPK-mTOR regulatory network in myoblasts.

Wound healing is a well-orchestrated process that typically recruits the immune and vascular systems to restore the structure and function of the injured tissue. Injuries to the enthesis, a hypocellular and avascular tissue, often result in fibrotic scar formation and loss of mechanical properties, thereby severely affecting musculoskeletal function and life quality. This raises questions about the healing capabilities of the enthesis. Here, we established an injury model to the Achilles entheses of neonatal mice to study the possibility that at an early age, the enthesis can heal more effectively. Histology and immunohistochemistry analyses revealed an atypical process that did not involve inflammation or angiogenesis. Instead, neonatal enthesis healing was mediated by secretion of collagen types I and II by resident cells, which formed a permanent hypocellular and avascular scar. Transmission electron microscopy showed that the cellular response to injury, including ER stress, autophagy and cell death, varied between the tendon and cartilage ends of the enthesis. Single-molecule in situ hybridization, immunostaining, and TUNEL assays verified these differences. Finally, gait analysis showed that these processes effectively restored function of the injured leg. Collectively, these findings reveal a novel healing mechanism in neonatal entheses, whereby local ECM secretion by resident cells forms an acellular ECM deposit in the absence of inflammation markers, allowing gait restoration. These insights into the healing mechanism of a complex transitional tissue may lead to new therapeutic strategies for adult enthesis injuries.

The uterus requires energy for sustained contractility during labor, to deliver the fetus and diminish the risk of postpartum hemorrhage. Our objective was to define energy requirements and assess metabolic flexibility in quiescent and contractile myometrial cells. Cells were treated with oxytocin to stimulate myometrial contractility. We found that myometrial cells rely on oxidative phosphorylation during quiescence and, when treated with oxytocin, can adapt to higher energy demands by shifting their energy production to glycolysis. Treatment with mitochondrial oxidation inhibitors revealed that in quiescent myometrial cells basal oxygen consumption rate decreased when treated with glucose oxidation inhibitor UK5099, but not the long chain fatty acid oxidation inhibitor etomoxir or the glutamine oxidation inhibitor BPTES. In oxytocin treated myometrial cells, this decrease was also observed upon BPTES treatment in addition to UK5099, suggesting that contractile myometrial cells can shift energy production from glucose to glutamine. Functionally, myometrial contractility was significantly reduced by UK5099 but not by etomoxir, further indicating dependence on glucose utilization.

Estradiol (E2), a sex steroid hormone molecule, plays a key role in regulating the actin and shape dynamics of cells in a multitude of normal and pathophysiological conditions. While cytoskeletal rearrangements, membrane dynamics, and cellular protrusions are intimately involved in cell motility and invasiveness, little is known about the impact of E2 on these processes in estrogen-dependent epithelial cells. In this study, we quantified the impact of E2 on cell shape and actin dynamics in 12Z human endometriotic epithelial cells transfected with LifeAct-GFP and observed with lattice lightsheet microscopy, a new imaging technique fast enough to capture 3D dynamics on second timescales. E2, when applied for 24 hours, significantly decreased cell circularity, solidity, and rate of change of circularity, indicating a transition to a more elongated and less variable morphology. 24-hour E2 treatment also induced the formation of large membrane protrusions reminiscent of invadopodia and led to a more disordered flow of actin within those protrusions. However, these effects were not seen after 15 minutes of E2 treatment, suggesting that longer-term signaling is required to drive these structural changes. Together, these results suggest that E2 modulates actin polymerization and membrane protrusion dynamics in endometriotic epithelial cells and may prime them for cell invasion. This work highlights a role for hormonal signaling in mediating cytoskeletal plasticity and migratory cell phenotypes.

The molecular interactions between the maternal environment and developing embryo that are key for early pregnancy success are known to be influenced by factors such as the metabolic status. We are, however, limited in our understanding of the mechanism by which these individual nutritional stressors alter endometrial function and the in utero environment for early pregnancy success. Here we report for the first time the use of endometrium-on-a-chip microfluidics approach to produce a multi-cellular endometrium in vitro, that is exposed to glucose and insulin concentrations associated with maternal metabolic stressors. Following isolation of endometrial cells (epithelial and stromal) from the uteri of non-pregnant cows in early-luteal phase (Day 4-7 approximately) epithelial cells were seeded into the upper chamber (4-6 104 cells/mL) and stromal cells seeded in the lower chamber (1.5-2 104 cells/mL). Three different concentration of glucose 1) 0.5 mM 2) 5.0 mM or 3) 50 mM or insulin 1) Vehicle, 2) 1 ng/mL or 3) 10 ng/mL were performed in the endometrial cells at a flow rate of 1{micro}L/min for 72 hr to mimic the rate of secretion in vivo. Quantitative differences in the transcriptomic response of the cells and the secreted proteome of in vitro-derived uterine luminal fluid (ULF) were determined by RNA-sequencing and TMT respectively. Changes in maternal glucose altered 21 and 191 protein coding genes in epithelial and stromal cells respectively (p<0.05). While there was a dose-dependent quantitative change in protein secretome (1 and 23 proteins). Insulin resulted in limited transcriptional changes including insulin-like binding proteins that were cell specific (5, 12, and 20) but altered the quantitative secretion of 196 proteins including those involved in extracellular matrix-receptor interaction and proteoglycan signaling in cancer. Collectively, these highlight the potential mechanism by which changes to maternal glucose and insulin alter uterine function.

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.

Meniscus injuries pose significant challenges in clinical settings, primarily due to the intrinsic heterogeneity of the tissue and the limited efficacy of current treatments. Endogenous cell migration is crucial for the healing process, yet the regulatory mechanisms of meniscus cell migration and its zonal dependency within the meniscus are not fully understood. Thus, this study investigates the role of epigenetic mechanisms in governing meniscus cell migration under inflammatory conditions, with a focus on their implications for injury healing and regeneration. Here, we discovered that a proinflammatory cytokine, TNF- treatment significantly impedes the migration speed of inner meniscus cells, while outer meniscus cells are unaffected, underscoring a zonal-dependent response within the meniscus. Our analysis identified distinct histone modification patterns and chromatin dynamics between inner and outer meniscus cells during migration, highlighting the necessity to consider these zonal-dependent properties in devising repair strategies. Specifically, we found that TNF- differentially influences histone modifications, particularly H3K27me3, between the two cell types. Transcriptome analysis further revealed that TNF- treatment induces substantial gene expression changes, with inner meniscus cells exhibiting more pronounced alterations than outer cells. Gene cluster analysis pointed to distinct responses in chromatin remodeling, extracellular matrix assembly, and wound healing processes between the zonal cell populations. Moreover, we identified potential therapeutic targets by employing existing epigenetic drugs, GSKJ4 (a histone demethylase inhibitor) and C646 (a histone acetyltransferase inhibitor), to successfully restore the migration speed of inner meniscus cells under inflammatory conditions. This highlights their potential utility in treating meniscus tear injuries. Overall, our findings elucidate the intricate interplay between epigenetic mechanisms and meniscus cell migration, along with its meniscus zonal dependency. This study provides insights into potential targets for enhancing meniscus repair and regeneration, which may lead to improved clinical outcomes for patients with meniscus injuries and osteoarthritis.

Injured tendons heal through the formation of a fibrovascular scar that has inferior mechanical properties compared to native tendon tissue. Reducing inflammation that occurs as a result of the injury could limit scar formation and improve functional recovery of tendons. Prostaglandin D2 (PGD2) plays an important role in promoting inflammation in some injury responses and chronic disease processes, and the inhibition of PGD2 has improved healing and reduced disease burden in animal models and early clinical trials. Based on these findings, we sought to determine the role of PGD2 signaling in the healing of injured tendon tissue. We tested the hypothesis that a potent and specific inhibitor of hematopoietic PGD synthase (HPGDS), GSK2894631A, would improve the recovery of tendons of adult male rats following an acute tenotomy and repair. To test this hypothesis, we performed a full-thickness plantaris tendon tenotomy followed by immediate repair and treated rats twice daily with either 0mg/kg, 2mg/kg, or 6mg/kg of GSK2894631A. Tendons were collected either 7 or 21 days after surgical repair, and mechanical properties of tendons were assessed along with RNA sequencing and histology. While there were some differences in gene expression across groups, the targeted inhibition of HPGDS did not impact the functional repair of tendons after injury as HPGDS expression was surprisingly low in injured tendons. These results indicate that PGD2 signaling does not appear to be important in modulating the repair of injured tendon tissue.

Desmosomes are junctional complexes that confer mechanical strength and enhance epithelial barrier function at mucosal surfaces by anchoring intermediate filaments to plasma membrane. While these roles are less explored in vaginal vs. cutaneous epithelium, we previously reported that treating mice with the progestin depot medroxyprogesterone acetate (DMPA) reduces vaginal epithelial levels of the desmosomal cadherins desmoglein-1 (DSG1) and desmocollin-1 (DSC1) and weakens vaginal epithelial barrier function. We also showed these effects were avoided by treating mice with DMPA and a conjugated equine estrogen vaginal cream. The current investigation further explored the effects of sex steroids on vaginal epithelial integrity, identifying ephrin-A3 (EFNA3) as a key regulator of desmosomal cadherin gene expression. We observed topical administration of recombinant EFNA3 (rEFNA3) promotes vaginal DSG1 expression in a biphasic dose-dependent manner and partially reverses the loss of vaginal epithelial barrier function induced by DMPA treatment. Consistent with this effect, morbidity and mortality elicited by genital herpes simplex virus type 2 infection were delayed, but not prevented, in mice administered DMPA and rEFNA3 vs. DMPA and vehicle. Together, these studies identify EFNA3 as an important regulator of desmosomal function in vaginal epithelium and improve current understanding of sex steroid-mediated mechanisms that control vaginal epithelial barrier function.

Intrauterine infection is a significant cause of preterm labor and neonatal morbidity and mortality. Ureaplasma parvum is the micro-organism most commonly isolated from cases of preterm birth and preterm premature rupture of membranes (pPROM). However, the mechanisms during the early stages of ascending reproductive tract infection that initiate maternal-fetal inflammatory pathways, preterm birth and pPROM remain poorly understood. To examine inflammation in fetal (chorioamnionic) membranes in response to Ureaplasma parvum infection, we utilized a novel in vivo non-human primate model of early choriodecidual infection. Eight chronically catheterized pregnant rhesus macaques underwent maternal-fetal catheterization surgery at 105-112 days gestation and choriodecidual inoculation with Ureaplasma parvum (105cfu/mL of a low passaged clinical isolate, serovar 1; n=4) or saline/sterile media (Controls; n=4) starting at 115-119 days gestation, repeated every 5 days until scheduled cesarean-section at 136-140d gestation (term=167d). The average inoculation to delivery interval was 21 days and Ureaplasma infection of the amniotic fluid was undetectable by culture and PCR in all animals. Inflammatory mediators in amniotic fluid (AF) were assessed by Luminex, ELISA and multiplex assays. RNA was extracted from the chorion and amnionic membranes for single gene analysis (qRT-PCR) and protein expression was determined by Western blot and immunohistochemistry. Our NHP model of choriodecidual Ureaplasma infection, representing an early-stage ascending reproductive tract infection without microbial invasion of the amniotic cavity, resulted in increased fetal membrane protein and gene expression of MMP-9 and PTGS2, but did not result in preterm labor (no increase in uterine contractility) or increased concentrations of amniotic fluid pro-inflammatory cytokines (IL-1{beta}, IL-6, IL-8, IL-18, TNF-). However, membrane expression of inflammasome sensor molecules, NLRP3, NLRC4, AIM2 and NOD2, and the adaptor protein ASC (PYCARD) gene expression were significantly increased in the Ureaplasma group when compared to non-infected controls. Gene expression of IL-1{beta}, IL-18, the IL-18R1 receptor, CASPASE-1 and pro-CASPASE-1 protein were also increased in the fetal membranes with Ureaplasma infection. Downstream inflammatory signaling genes MYD88 was also significantly upregulated in both the amnion and chorion, along with a significant increase in NFKB in the chorion. These results demonstrate that even at the early stages of ascending reproductive tract Ureaplasma infection, activation of inflammasome complexes and pathways associated with degradation of chorioamnionic membrane integrity are present. This study therefore provides experimental evidence for the importance of the early stages of ascending Ureaplasma infection in initiating processes of pPROM and preterm labor. These findings have implications for the identification of intrauterine inflammation before microbes are detectable in the amniotic fluid (sterile inflammation) and the timing of potential treatments for preterm labor and fetal injury caused by intrauterine infection.

Tjp1 + is considered a crucial protein involved in the stepwise assembly of tight junctions (TJs) between compaction and blastocoel cavitation in early development. In this study, we investigated the specific role of Tjp1 + in TJ formation by employing an alternative splicing-specific knockdown of the Tjp1 + exon. To deplete Tjp1 + expression, we used siRNA targeting RNA-binding protein 47 (Rbm47), which induces the inclusion of the + exon in Tjp1 mRNA. The knockdown resulted in approximately 85% reduction in Rbm47 mRNA levels and 75% reduction in Tjp1 + mRNA levels in blastocysts. Surprisingly, despite this knockdown, blastocyst development and TJ permeability of trophectoderm were unaffected. Additionally, we observed an interaction between Tjp1 - and Ocln in Rbm47 knockdown blastocysts, suggesting a compensatory role of Tjp1 -. Overall, our findings indicate that Tjp1 + is not essential for the stepwise assembly of TJs and the completion of TJ biogenesis during blastocyst development in mice although a minimal amount of remaining Tjp1 + is sufficient for TJs assembly. Summary statementSelective loss of Tjp1 + mediated by Rbm47 knockdown did affect mouse blastocyst development, suggesting that Tjp1 + may not be crucial for stepwise TJs assembly during blastocyst development

Endometrial regeneration is essential for reproductive cycles and pregnancies, allowing the endometrium to undergo repair and renewal after menstruation and parturition. Epithelial cells lining the uterine cavity are shed during each cycle, although remnant luminal and glandular epithelial cells can regenerate the lumen lining. It is presumed that adult stem/progenitor cells in the uterine stroma also contribute to this regeneration. However, the specific cell type(s) and the underlying mechanisms have not been determined. Herein, we use genetic lineage tracing assays in mice to identify Nestin+ perivascular cells as active contributors to epithelial regeneration. Notch signaling maintains Nestin+ perivascular cells in a quiescent state but these cells re-enter the cell cycle and differentiate into epithelial cells via estrogen-stimulated suppression of Notch signaling dependent on estrogen receptor alpha (ER/ESR1). These findings demonstrate that perivascular cells support epithelial regeneration and reveal a mechanism regulating the quiescence and activation of uterine perivascular cells.

Altered mitochondrial structure and function are implicated in the functional decline of skeletal muscle. Numerous cytoskeletal proteins have been reported to affect mitochondrial homeostasis, but this complex network is still being unraveled. Here, we investigated alterations to mitochondrial structure and function in mice lacking the cytoskeletal adapter protein, Xin. Xin deficient (Xin-/-) and wild-type (WT) littermate mice were fed a chow or high-fat diet (HFD; 60% kcal fat) for 8 weeks before high-resolution respirometry, histology, electron microscopy and Western blot analyses of their skeletal muscles were conducted. Immuno-electron microscopy and immunofluorescence staining indicates that Xin is present in the mitochondria and peri-mitochondrial areas, as well as the myoplasm. Intermyofibrillar mitochondria in chow-fed Xin-/- mice were notably different from WT; frequently spanning a whole sarcomere and/or swollen in appearance with abnormal cristae. Succinate Dehydrogenase and Cytochrome Oxidase IV (COX) activity staining indicated greater evidence of mitochondrial enzyme activity in Xin-/- mice. HFD did not result in a difference between cohorts with respect to body mass gains or glucose handling. However, electron microscopy revealed significantly greater mitochondrial density ([~]2.1-fold) with evident structural abnormalities (swelling, reduced cristae density) in Xin-/- mice. Complex I and II-supported respiration were not different between groups per mg muscle, but when made relative to mitochondrial density, were significantly lower in Xin-/- muscles. Western blotting of fusion, fission, and autophagy proteins revealed no differences between groups. These results provide the first evidence for a role of Xin in maintaining mitochondrial morphology and function but not in regulating mitochondrial dynamics.

Epithelial cells lining the lumen of organs have an apical-basal polarity, and mechanical cell contacts are located in the lateral and basal regions. During early pregnancy in rodents, the luminal space of the uterus is closed, and opposing epithelial cells attach to each other at apical-apical surfaces. Here, we show that cells are mechanically coupled at the apical cell-cell contacts. The apical plasma membrane was intricately intertwined between opposing cells. Extracellular matrix and tight junction molecules were localized at apical contacts. Claudin inhibition resulted in impaired luminal closure, suggesting a functional requirement of claudins for the establishment of transapical coupling. The present results demonstrate a mechanical cell-cell interaction that occurs between apical-apical surfaces in addition to lateral and basal cell junctions. One Sentence SummariesTransapical coupling of epithelial cells mediated by extracellular matrix and tight junction molecules