The FASEB Journal

Maternal obesity and viral infection induce placental inflammation, but how their co-exposure influence fetoplacental development remains unclear. We hypothesized that maternal high fat (HF) diet and viral infection would independently induce placental inflammation and lipid peroxidation, reduce antioxidant defence, and cellular turnover. Further, HF diet would compromise placental capacity to adapt to infection. Female C57BL/6J mice were fed a control (CON) or 62% HF diet six weeks before and throughout pregnancy and injected with poly(I:C) (viral mimic) or vehicle (VEH) 24h before sacrifice at gestational days (GD) 12.5, 15.5, and 18.5 (n=5-8/group/GD). Placental inflammasome (NLRP3), oxidative stress (4-HNE), antioxidant defence (GPx-4), and cellular proliferation-to-death ratio (Ki-67, Caspase-3) were assessed by immunohistochemistry, and mRNA expression of Tlr3, Irf3, Tlr4, Tirap, and Il-1{beta} were measured by qPCR. Data were analysed by linear mixed models (p[≤]0.05). At GD12.5, infection was associated with increased Tlr3 mRNA and immunoreactive (ir)-4-HNE, and reduced ir-GPx-4 expression in the placental labyrinth zone (LZ). By GD15.5, HF diet was associated with increased ir-NLRP3 in both LZ and junctional zones (JZ). Exposure to infection alone and co-exposure to HF diet and infection further increased LZ ir-NLRP3. At GD18.5, HF diet was associated with increased Tirap and Il-1{beta} mRNA expression, ir-4-HNE in the JZ and ir-Caspase-3 in the LZ. Maternal HF diet and infection exert distinct effects on the placenta across gestation, suggesting that maternal overnutrition might reduce the placentas capacity to handle adverse exposures, which may increase susceptibility to poor fetal outcomes.

Physiological load is vital for maintaining tendon homeostasis, preserving the organized extracellular matrix that enables tendons to withstand extreme forces. Although tenocytes are regarded as the primary regulators of extracellular matrix production, precisely how cells facilitate the maintenance of homeostasis in response to physiological load is poorly understood. Here, we used Voluntary Wheel Running (VWR) as a model of physiological load to delineate the specific cellular contributions to mouse Achilles tendon homeostasis. Eight weeks of VWR led to a smaller cross-sectional area, increased mechanical and material properties at the midsubstance, which corresponded to a decreased proportion of small (0-60 nm) collagen fibrils and an increased proportion of larger (100-60 nm) collagen fibrils compared to sedentary controls. Using Visium HD spatial transcriptomics, we identified region-specific cell clusters (insertion vs. midsubstance). In response to physiological load, cells in the insertion and midsubstance upregulate distinct genes that reinforce the fibrocartilage interface and collagen-rich tendon matrix, respectively. Notably, Clu, Myoc, and Ccdc80 were upregulated with VWR in the midsubstance, with previously uncharacterized roles in tendon homeostasis. Together, our findings suggest that in response to physiological load, tendon cells maintain homeostasis by region-specific responses. Given that insertional and midsubstance tendinopathy is function-limiting and painful, defining the region-specific cellular responses will be key to advancing therapeutic prospects for tendon health. New and NoteworthyThis study is the first spatially rigorous characterization of the tendon response to physiological load using a Voluntary Wheel Running (VWR) model. VWR led to smaller, stronger, but not stiffer tendons at the midsubstance compared to sedentary controls. This corresponded with significant decreased proportion of small collagen fibrils and a shift toward an increased proportion of large collagen fibrils. Using Visium HD spatial transcriptomics, we identified region-specific transcriptional responses to physiological load that maintain homeostasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/722285v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@13aa717org.highwire.dtl.DTLVardef@1afe3f1org.highwire.dtl.DTLVardef@9e1137org.highwire.dtl.DTLVardef@17c47bc_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cholesterol elimination in mammals depends largely on the biliary secretion of cholesterol and its conversion to bile acids, followed by their fecal loss. Human studies suggest an association between blood vitamin D levels and blood cholesterol; however, the mechanistic impact of sustained elevation of 1,25(OH)2D3 (active vitamin D) on cholesterol flux remains unclear. Here, we used two complementary mouse models--a genetic model with chronically elevated plasma 1,25(OH)2D3 (-klotho KO mice) and a pharmacological model of repeated 1,25(OH)2D3 administration in wild-type mice--to define the mechanism by which 1.25(OH)2D3 regulates the hepatic-intestinal programs controlling cholesterol elimination. -klotho KO mice showed increased fecal excretion of both cholesterol and total bile acids. Hepatically, Sr-b1, Abcg5/Abcg8, Abca1, Cyp7a1, and Mrp2 transcriptions were increased, whereas Cyp27a1 and Bsep was unchanged. Duodenal Npc1l1 was reduced, and ileal Asbt showed a decreasing trend. In the administration model, fecal bile acid levels increased by day 3, consistent with the induction of hepatic Mrp2 expression from day 3. Bsep exhibited a biphasic change, enhanced at early phase and downregulated to basal levels later and Asbt was unchanged. Increased fecal cholesterol emerged later (day 15), accompanied by late-phase induction of Abcg5/Abcg8 and suppression of Npc1l1. Together, we propose that sustained elevation of 1.25(OH)2D3 is associated with coordinated hepatic and intestinal transcriptional remodeling that promotes cholesterol disposal, with an early increase in fecal bile acid loss preceding the enhanced fecal cholesterol excretion.

The ketogenic diet (KD) promotes ketone body synthesis and has been used as an effective treatment for disorders such as epilepsy. Although elevated ketone bodies, including {beta}-hydroxybutyrate ({beta}HB) and acetoacetate, are thought to meditate the beneficial effects of the KD, the mechanisms underlying their metabolic actions remain incompletely understood. In this study, we focused on GPR109A, a receptor for {beta}HB with an unclear role in metabolic homeostasis. We employed KD and fasting models to examine metabolic changes under two distinct ketogenic conditions. Under KD conditions, Gpr109a-/- mice exhibited increased hepatic lipid accumulation, and subsequent hepatic inflammation and fibrosis. However, Gpr109a deletion did not exacerbate hepatic lipid accumulation or inflammation during short-term fasting, suggesting that GPR109A-mediated liver protection is specific to KD-induced metabolic stress rather than under fasting conditions. Mechanistic analysis revealed that GPR109A protects the liver from inflammation by maintaining intestinal barrier integrity. These findings highlight the novel protective mechanism of GPR109A, via the gut-liver axis, to sustain metabolic homeostasis during the KD. This study provides valuable insights into the physiological effects of ketone bodies.

Statins, widely used cholesterol-lowering drugs, inhibit the mevalonate pathway and reduce coenzyme Q (CoQ) biosynthesis, potentially impairing mitochondrial function. Because astrocytes are essential for maintaining brain redox homeostasis, statin-induced mitochondrial dysfunction in these cells may contribute to CNS pathology. We examined the effects of a six-day statin exposure on mitochondrial bioenergetics in rat astrocytes, focusing on mitochondrial CoQ (mtCoQ) deficiency. Treatment with 200 nM atorvastatin or simvastatin decreased the total mtCoQ pool (mtCoQ9 + mtCoQ10) by 30-35% and decreased the antioxidant pool mtCoQH2 by 40%, whereas the levels of mitochondrial antioxidant proteins, including superoxide dismutase 2 and uncoupling proteins, remained unchanged. Mitochondria of statin-treated astrocytes showed decreased respiratory activity, membrane potential, and ATP synthesis, and increased mtCoQ reduction leading to increased H2O2 production during the oxidation of complex I (CI) and CII substrates. Statin treatment also altered the organization of the respiratory chain, leading to a downregulation of the CI+CIII2+CIV and CIII2+CIV supercomplexes and decreased protein levels and activity of all respiratory chain complexes. Furthermore, a decrease in cytochrome a + a3 content was accompanied by a reduction in the maximum activity of CIV. CoQ10 supplementation elevated mtCoQ levels, restored respiratory function, and decreased H2O2 production in the mitochondria of statin-treated astrocytes. Prolonged statin exposure alters mtCoQ redox homeostasis and impairs mitochondrial bioenergetic function in astrocytes. CoQ10 supplementation attenuates these changes, supporting its potential role in protecting astrocyte mitochondria from statin-induced dysfunction.

In eutherian mammals, blastocyst implantation is often associated with a quasi-inflammatory reaction in the endometrium, which is resolved with the establishment of the definitive placenta. This is understandable in the case of invasive placentation, since implantation entails a nidatory injury to the maternal tissue due to the invading blastocyst. Quasi-inflammatory processes have also been documented in pregnant pigs, even though the blastocyst only attaches to, rather than invades into, the endometrium of the uterus. In this study, we asked what processes in early porcine pregnancy lead to the resolution of attachment-associated inflammation. In generic wound healing the transition from a pro- to an anti-inflammatory state is caused by a corresponding transition from M1 to M2 polarized macrophages following efferocytosis by macrophages of apoptotic neutrophils. In order to determine whether this scenario applies to the pregnancy-related resolution of inflammation in the porcine uterus, we produced a series of bulk transcriptome samples spanning days (D) 13 to 25 of gestation. This time span corresponds to the transition from pre- to post-attachment stages of pregnancy. We found slower changes in the transcriptome between D20 and D25 than prior to D20, suggesting a turning point in pregnancy-related reprogramming. The turning point at D20 corresponds to the time of firm attachment of trophectoderm to uterine luminal epithelium and the cessation of IFNG signaling from the blastocyst. This transition coincides with increased expression of RNAs of genes implicated in resolution of inflammation and M2 polarization such as ARG1, MRC1/CD206, CD86, TGFb1 and IL10, as well as a significant increase in expression of HGPD, the enzyme that metabolizes prostaglandins. While immunoreactivity for ARG1 was found in putative macrophages in the sub-epithelial stratum compactum, other markers of M2 polarized macrophages were localized to non-immune cells: MRC1 was found on fibroblast-like stromal cells, CD86 on trophoblast cells, and IL10 in luminal and glandular epithelia. These results suggest that intrauterine immune regulation is decoupled from that of the rest of the body by engaging non-immune cell types as anti-inflammatory mediators during the peri-attachment period of pregnancy.

Maternal heat stress (HS) is an emerging risk factor for adverse pregnancy outcomes, yet how gestational heat exposure causes persistent placental dysfunction remains unclear. In the present study, we established a murine HS model (38.5{degrees}C, 2.5 h/day, E0-E12.5) followed by thermal recovery to E17.5. HS reduced fetal weight during early gestation and caused persistent fetal growth restriction after recovery, despite partial placental weight restoration. Histological analyses revealed early reductions in the junctional and labyrinth zones, followed by sustained labyrinthine deficiency and compensatory junctional zone expansion. Consistently, HS impaired placental vascularization, with reduced vessel length and area, decreased CD31 and -SMA abundance, and altered angiogenesis-related gene expression. HS also triggered oxidative stress, weakened antioxidant capacity, disrupted anti-inflammatory signaling, reduced tight junction protein expression, and compromised barrier integrity. Mechanistically, HS induced excessive endoplasmic reticulum stress, accompanied by increased CHOP, phosphorylated ERK, and cleaved caspase-3. In conclusion, our data unveil a heat-induced placental insufficiency program that restricts fetal growth through vascular, redox, barrier, and ERS-MAPK-apoptotic remodeling. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/730306v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@177be8forg.highwire.dtl.DTLVardef@7faa52org.highwire.dtl.DTLVardef@1870971org.highwire.dtl.DTLVardef@7d1705_HPS_FORMAT_FIGEXP M_FIG C_FIG

Ovarian stimulation is widely used in assisted reproductive technologies, yet its effects on uterine architecture and embryo implantation remain poorly understood. Using a mouse model, we show that ovarian stimulation or superovulation disrupts pre-implantation luminal epithelial folding and induces aberrant smooth muscle structure and contractile function. These structural defects result in embryo trapping within aberrant longitudinal folds, impaired implantation chamber formation, misalignment of the embryo-uterine axis, and subsequent embryo loss. These ovarian stimulation effects were reversible after rest and restoration of normal estrus cycling. Transcriptomic analysis suggests widespread disruption in the stroma and immune compartments of the stimulated uteri. Pathway analysis revealed significant disruption of stromal extracellular matrix and enhanced probability of stroma-immune communication via collagen signaling. Platelet derived growth factor receptor A (PDGFRA) expression was elevated in both the stroma and smooth muscle of the stimulated uteri. Short-term pharmacological inhibition of PDGFRA in the stimulated uteri prior to implantation fully restored epithelial fold transition and implantation chamber formation and partially restored smooth muscle architecture and contractility. Importantly, PDGFRA protein was also elevated in endometrial biopsies from women undergoing ovarian stimulation when compared to natural cycle biopsies. Together, this study establishes muscle contractions and stromal and smooth muscle PDGFRA signaling as novel non-cell autonomous regulators of uterine epithelial architecture critical for embryo implantation.

ObjectiveInvestigate how Ca2+/calmodulin dependent protein kinase kinase 2 (CaMKK2) orchestrates a catabolic shift in chondrocytes during early osteoarthritis (OA). MethodsCartilage, osteochondral plugs and chondrocytes were collected from patients undergoing total hip arthroplasty or non-OA donors. SOX9 levels were assessed via immunoblotting or immunohistochemistry (IHC). Sox9 levels were also assessed by IHC in knee joints from wild-type (WT) and Camkk2-/- mice that underwent sham or destabilization of medial meniscus (DMM), with or without STO-609 (0.033 mg/kg) treatment. Co-immunoprecipitation followed by mass spectrometry was performed to identify CaMKK2 interacting proteins in chondrocytes. Kinase assays were analyzed by immunoblotting and phosphosites identified by mass spectrometry. Proteasome function was assessed in murine and human chondrocytes lacking or expressing kinase-active or kinase-inactive CaMKK2. ResultsInhibition or loss of CaMKK2 increased SOX9, whereas the expression of kinase-active, not inactive, CaMKK2 reduced Sox9 in human and mouse OA cartilage. Proteomic analysis of CaMKK2 immunoprecipitates revealed the presence of ubiquitin E3 ligase Ubr4 and the 19S proteasome regulatory particle (RP). CaMKK2 kinase activity was dispensable for its interactions with Ubr4, 19S RP, and Sox9-ubiquitin conjugates, and kinase-inactive CaMKK2 attenuated Sox9 degradation in chondrocytes. Further, CaMKK2 phosphorylated the 19S RP ATPase Psmc5 on Ser136, and an intact kinase increased proteasome activity in chondrocytes. ConclusionsOur findings identify CaMKK2 as a dual-function regulator of chondrocyte UPS with a scaffolding role to assemble UPSUbr4-19S RP around polyubiquitinated proteins such as Sox9, and a catalytic role to enhance proteasome function, potentially through Psmc5 phosphorylation, thereby linking chondrocyte inflammatory signaling to Sox9 degradation and cartilage degeneration.

Iron absorption in the small intestine has classically been described by the duodenal DMT1/FPN1 pathway for inorganic non-heme iron, yet emerging evidence suggests that chemically distinct iron forms may use region-specific routes. Nicotianamine (NA), a plant-derived metal chelator, can form NA-iron (NA-Fe) complexes and has been proposed to support intestinal iron absorption through amino acid transporter pathways. However, direct comparisons of transepithelial transfer of inorganic iron and NA-Fe across defined small intestinal regions under controlled epithelial conditions remain limited. Here, we established region-specific 2D epithelial monolayers derived from duodenal and proximal jejunal crypt organoids from male ICR mice cultured on Transwell inserts. Transcriptomic profiling indicated partial retention of regional identity, and barrier integrity was confirmed by junctional marker localization, transepithelial electrical resistance, and low paracellular permeability. We then examined expression and polarized localization of candidate transporters for inorganic iron (Dmt1/Fpn1) and NA-Fe (Pat1/Lat2). Finally, we quantified transepithelial transport using apical loading of isotope-labeled iron (55Fe) or NA-55Fe and measured radioactivity appearing in the basolateral compartment as the primary readout of transepithelial flux. Basolateral appearance of inorganic 55Fe was comparable between duodenum- and proximal jejunum-derived monolayers, whereas NA-55Fe exhibited significantly greater basolateral appearance in proximal jejunum-derived monolayers. These findings demonstrate that organoid derived, region-specific monolayers provide a tractable epithelial platform to evaluate iron form-dependent, region-specific transepithelial transfer and to enable further mechanistic dissection of NA-Fe transport. NEW & NOTEWORTHYNon-heme iron absorption may depend on iron chemical form and intestinal region, but direct epithelial comparisons are scarce. We established duodenum and proximal jejunum derived murine intestinal organoid monolayers on Transwells and quantified transepithelial flux using isotope-labeled iron. Inorganic 55Fe showed no clear regional difference, whereas NA-55Fe displayed greater basolateral appearance in proximal jejunum-derived monolayers. This platform enables mechanistic studies of NA-iron complex transport.

CCCTC-binding factor (CTCF) is frequently mutated in endometrial cancer, resulting in genetic haploinsufficiency that contributes to tumour progression. We previously showed that depletion of CTCF disrupted cell polarity in KLE endometrial cancer spheroids; however, the implications for gene dysregulation and endometrial cancer pathophysiology remains poorly understood. ZNF185, an actin-associated and LIM domain-containing protein involved in cytoskeletal remodelling, was identified as a dysregulated target following CTCF haploinsufficiency. In this study, shRNA-mediated knockdown of CTCF was used to model haploinsufficiency in endometrial cancer cells, leading to the identification of a previously unrecognised isoform of ZNF185, named ZNF185B. Unlike the full-length protein, ZNF185B lacked co-localisation with F-actin and exhibited a diffuse cytoplasmic distribution, and ZNF185B was significantly upregulated in CTCF-depleted endometrial cancer cells and in an auxin-inducible degron model in a dose-dependent manner. Functional studies demonstrated that depletion of ZNF185 expression reduced endometrial cancer cell proliferation and clonogenic potential. Together, these findings identify ZNF185B as a novel isoform negatively regulated by CTCF protein dosage and establish ZNF185 as a requirement for endometrial cancer cell proliferation. Our results suggest that dysregulated ZNF185 expression is a crucial downstream consequence of CTCF haploinsufficiency and may contribute to tumour progression in endometrial cancer.

Sedentary behavior promotes chronic low-grade inflammation in adipose tissue, contributing to metabolic dysfunction and insulin resistance. High-intensity interval training (HIIT) is a time-efficient exercise strategy with potent anti-inflammatory and metabolic benefits; however, its effects on adipose tissue inflammatory signaling and microRNA (miRNA) regulation remain incompletely understood. This study investigated the effects of eight weeks of HIIT on inflammatory and epigenetic markers in interscapular white adipose tissue (iWAT) of male Wistar rats. Fourteen rats were randomly assigned to either a sedentary (SED; n = 7) or HIIT (n = 7) group. The HIIT protocol consisted of treadmill running five days per week for eight weeks. Body weight and iWAT mass were assessed, and molecular adaptations were evaluated at multiple regulatory levels using RT-qPCR for mRNA targets (NLRP3, TNF-, PPAR-{gamma}, and IL-10) and miRNAs (miR-21 and miR-30d-5p), while protein levels of NLRP3 and PPAR-{gamma} were assessed using Western blotting. Compared with the SED group, HIIT significantly reduced body weight (p < 0.001) and iWAT mass (p = 0.002). Furthermore, HIIT downregulated the expression of pro-inflammatory mediators, including NLRP3 (gene: p = 0.001; protein: p < 0.001) and TNF- (p = 0.025), while upregulating anti-inflammatory regulators PPAR-{gamma} (gene: p = 0.026; protein: p = 0.020) and IL-10 (p = 0.010). In parallel, inflammation-associated miRNAs, including miR-21 (p = 0.004) and miR-30d-5p (p = 0.002), were markedly downregulated. These coordinated transcriptional, post-transcriptional, and translational adaptations suggest that HIIT attenuates adipose tissue inflammation and promotes a favorable immunometabolic phenotype through integrated molecular and epigenetic mechanisms.

Regular exercise induces adaptations in skeletal muscle and other organ systems to improve physical performance and overall health. Exercise results in phosphorylation of 5 AMP-activated protein kinase (AMPK) at threonine 172 (T172) of the 2 subunit; however, the role of this activation in cellular and functional adaptations has not been elucidated. To this end, we subjected non-activatable Ampk2(T172A) knock-in (KI) adult mice and wild-type (WT) littermates to 4 weeks of voluntary wheel running (VWR). Exercise training led to significant improvements in endurance capacity, maximal oxygen consumption ([Formula]O2max), and glucose tolerance, as well as skeletal muscle IIb-to-IIa fiber type shift in both WT and KI mice. Contrastingly, VWR resulted in increased mitochondrial OxPhos protein expression, mitochondrial volume density, and capillary density in skeletal muscle of WT but not KI mice. Exercise-induced improvements of mitochondrial respiration and conductance revealed by high-resolution respirometry of isolated mitochondria were blunted in KI mice. Therefore, for the first time, we reveal that AMPK2 T172 activation is required for exercise training-induced mitochondrial biogenesis, improvement of mitochondrial respiratory function, and angiogenesis in skeletal muscle, but that these adaptations are not solely responsible for improved [Formula]O2max and exercise endurance capacity. Significance StatementExercise is the most effective lifestyle intervention for promoting health and preventing chronic diseases through adaptive changes in skeletal muscle and many other tissues/organs. AMPK is an energy sensor and signaling regulator for exercise-induced skeletal muscle adaptation, yet its functional role and the impact on exercise capacity have been studied in mouse genetic models wherein protein stoichiometry is disrupted. Using non-activatable Ampk2(T172A) knock-in mice, we ascertained that AMPK2 activation via T172 phosphorylation is required for endurance training-induced mitochondrial and angiogenic adaptations in skeletal muscle. Importantly, these adaptations are not required for improved exercise capacity, challenging the prevailing concept that increased mitochondrial content and function and microvasculature are the sole driving factors for the performance gains with endurance training.

NKCC2, localized to the apical membrane of thick ascending limb epithelial cells, is essential for renal salt handling and systemic electrolyte homeostasis. NKCC2 undergoes extensive ubiquitylation, with the E3 protein ligase Nedd4-2 implicated as a key regulator. However, progress has been limited by challenges expressing NKCC2 in mammalian cell lines, hindering mechanistic studies of NKCC2 ubiquitylation. Therefore, the aims of this study were to develop a mammalian cell model enabling mechanistic investigations of NKCC2 ubiquitylation, including the role of Nedd4-2 and the functional consequences of site-specific modification. A tetracycline-inducible MDCKI cell line was generated expressing human NKCC2 and used to assess Nedd4-2-dependent and site-specific ubiquitylation of NKCC2 using biochemical, imaging, and functional assays. The MDCKI cell line demonstrated stable, inducible expression of full-length human NKCC2. In this cell line, mutating the ubiquitylation site at K871 increased membrane abundance and uptake activity, without altering internalization rates. Nedd4-2 co-immunoprecipitated with NKCC2, and Nedd4-2 deletion increased total, but not membrane NKCC2 abundance. In summary, ubiquitylation on NKCC2 at K871 plays a key role in controlling NKCC2 membrane localization and thus function. Although Nedd4-2 can modulate NKCC2 abundance, it is not involved in NKCC2 trafficking. We conclude that the generated cell line provides a robust model for mechanistic studies of NKCC2 and will aid studies examining posttranslational regulation of NKCC2.

We investigated effects of three aerobic exercise interventions, varying in amount and intensity with durations of 8-9-months on small RNA (smRNA) expression and regulatory pathways in skeletal muscle and plasma from 120 participants. Using untargeted smRNA sequencing focused on miRNAs and piRNAs, adjusting for demographics and bodyweight, we identified 124 muscle smRNAs altered by exercise amount and 15 by intensity, and 47 plasma smRNAs altered by intensity and one by amount. These smRNAs were enriched in metabolic, transcriptional, translational, and cell cycle pathways. Exercise-induced changes in several smRNAs-six from muscle and five from plasma-and exercise-induced reduction in body weight, aligned with improvement in insulin sensitivity (p<0.05). These findings demonstrate tissue-specific regulation of smRNAs by exercise and identify potential candidates for exercise mimetics to modulate muscle insulin sensitivity.

Vitamin D signaling has recognized roles in female reproductive physiology, but its effects at the chromatin level in endometrial stromal cells are still unclear. Here, we investigated how the active form of vitamin D, 1,25-dihydroxyvitamin D3, or calcitriol, influences the accessible chromatin landscape of human endometrial stromal cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed on T-HESCs treated with either a vehicle or 1,25(OH)2D3. Ligand treatment increased overall chromatin accessibility, shown by higher ATAC-seq signal intensity, while causing only minor changes in the total number of called peaks. Peak annotation revealed that accessible regions were spread across both promoter-proximal and distal genomic areas. Integrating this data with CUT&RUN and RNA sequencing showed that most vitamin D-responsive cistromic modifications and transcripts were linked to nearby open chromatin, though fewer were associated with regions that were significantly differentially accessible. These results suggest that 1,25(OH)2D3-dependent transcription mainly occurs within a permissive, pre-accessible chromatin environment. This study offers new evidence that active vitamin D influences the epigenomic landscape of human endometrial stromal cells, establishing the chromatin-based molecular response to a chemically-defined VDR ligand, 1,25(OH)2D3, relevant to stromal differentiation and preparation for decidualization. HighlightsO_LIFirst evidence suggesting the direct impact of active vitamin D, 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, enhanced the signal intensity of chromatin accessibility in human endometrial stromal cells C_LIO_LIMost accessible chromatin regions were shared between vehicle and ligand-treated human endometrial stromal cells C_LIO_LI1,25(OH)2D3-responsive transcription occurs largely within pre-accessible chromatin in human endometrial stromal cells C_LIO_LIAssay for transposase-accessible chromatin sequencing (ATAC-seq) defines a chromatin-level pharmacologic response to a chemically defined VDR ligand in human endometrial stromal cells C_LI

Circadian regulation of proteostasis, a key determinant of muscle health, remains poorly understood. Here, we identified DNAJB6, an Hsp40 (DnaJ) co-chaperone, as a substrate of the circadian E3 ligase FBXL21. FBXL21 mediated the ubiquitination-dependent proteasomal degradation of both DNAJB6 and its client proteins including Desmin; causative mutations of DNAJB6 in myopathies, however, rendered resistance to FBXL21-directed degradation. Fbxl21 KO C2C12 cells displayed aberrant accumulation of Desmin, and showed aggravated cytoplasmic accumulation of TDP-43, another DNAJB6 client protein, in heat shock response. Under timed exercise as a physiological stressor, WT mice displayed robust diurnal rhythms in the levels of stress granule markers (G3BP1 and FUS) and TDP-43 as a function of exercise timing. In contrast, the Fbxl21 hypomorph Psttm mutant mice showed elevated expression of these proteins without exercise, which was exacerbated under exercise-induced stress conditions; importantly, these abnormalities were rescued by skeletal muscle-specific FBXL21 expression. Our study elucidates a novel diurnal regulatory mechanism of skeletal muscle proteostasis via FBXL21 as a chaperone-linked E3 ligase, highlighting the FBXL21-DNAJB6 axis as a potential therapeutic target for myopathies.

BackgroundDiscrete subaortic stenosis (DSS) is a pediatric cardiovascular disease marked by fibrotic growth within the left ventricular outflow tract (LVOT), leading to severe complications, including left ventricular hypertrophy, aortic regurgitation, and arrhythmias. Despite surgical intervention, a 20-30% recurrence rate suggests a complex underlying pathophysiology. Elevated flow and resultant hemodynamic shear stress within the LVOT are key factors in DSS development. While effects of shear stress on endothelial cells have been studied, the impact on macrophages and their interactions with endothelial cells remains unclear. MethodsIn this study, human monocyte-derived macrophages (MDMs) and human aortic endothelial cells (HAECs) were subjected to shear using a cone-and-plate viscometer. Cellular crosstalk was evaluated through conditioned media (CM) transfers. Gene expression, permeability and chemotaxis assays, immunofluorescent staining, and ELISAs assessed cellular responses. ResultsMDMs exposed to shear stress exhibited a pro-inflammatory response with upregulated TNF and CXCL8 genes. HAECs exposed to MDM-CM showed increased expression of inflammatory markers (VCAM-1, ICAM-1) and decreased VE-Cadherin and CD31, indicating increased permeability. Permeability assays confirmed that HAECs became more permeable when exposed to MDM-CM. Chemotaxis assays showed time-dependent monocyte migration in both MDM-CM and HAEC-CM. Immunofluorescent staining revealed diminished VE-Cadherin and CD31 in HAECs exposed to MDM-CM. ConclusionsOverall, pathological shear stress induced macrophages to secrete factors that increased endothelial permeability and perpetuated an inflammatory response. This interaction likely exacerbates fibrosis in DSS, promoting recurrence post-surgery. Understanding these mechanisms opens potential therapeutic avenues targeting inflammatory crosstalk between macrophages and endothelial cells, which could mitigate fibrosis and improve patient outcomes.

PurposeBacterial vaginosis (BV) is associated with disruption of the vaginal microbiome and extracellular matrix (ECM) remodeling, yet the contribution of host proteases to this process remains unclear. This study investigated whether expression and activity of cathepsins K, L, S, and V differ by BV diagnosis and community state type (CST). We hypothesized that BV and BV associated CSTs would exhibit increased expression and activity of collagen and elastin-degrading cathepsins. MethodsVaginal fluid samples were collected and classified by BV diagnosis and CST. Cathepsin expression was evaluated by Western blotting to distinguish inactive and active enzyme forms. Proteolytic activity was assessed using multiplex cathepsin zymography. Statistical analyses compared cathepsin expression and activity across diagnoses and CSTs. Principal component analysis and linear regression were performed to assess associations between cathepsin activity, microbial diversity, and CST. ResultsProcathepsin K expression was significantly increased in BV-positive and CST IV samples, while total cathepsin L expression was significantly elevated in samples with Nugent-intermediate scores. Cathepsins S and V showed variation in inactive and active forms in Nugent-intermediate and CST III samples. In contrast, total cathepsin activity, including cathepsins K and V, did not significantly differ across BV diagnoses or CSTs. Overall, cathepsin activity varied between individuals rather than by clinical classification. ConclusionsCathepsin expression and maturation state differ by microbiome composition, suggesting that the vaginal microbiome may regulate post-translational processing of cathepsins. As a result, cathepsin activity appears to be regulated at the individual level rather than strictly by BV diagnosis or CST. These findings link vaginal microbiome composition to ECM remodeling and potential adverse reproductive outcomes.

BackgroundPregnancy and lactation impose substantial demands on maternal calcium homeostasis, leading to pronounced skeletal remodeling during lactation followed by recovery after weaning. Although bone mass is largely returned at the tissue level after weaning, it remains unclear whether osteocyte-level remodeling exhibits a similarly reversible pattern. Osteocytes regulate mineral mobilization through perilacunar/canalicular remodeling (PLR), which is elevated during lactation. However, its spatial and temporal regulation in response to reproduction remains poorly defined. Objective/HypothesisThis study aims to determine whether PLR-regulated lacunar remodeling during reproduction varies with osteocyte location and relative age. We hypothesized that osteocyte PLR-mediated lacunar change is spatially heterogeneous during lactation, varies with osteocyte location and relative age, and may persist after weaning. MethodsFemale wild-type (WT) and osteocyte-specific PTH/PTHrP receptor knockout (cKO) mice were studied across reproductive stages. Longitudinal in vivo DXA and CT were used to assess skeletal changes. Sequential fluorochrome labeling tracked mineral dynamics and defined osteocyte relative age. Osteocyte PLR activity was evaluated by MMP13 immunohistochemistry. Lacunar-canalicular structure (LCS) was assessed using Ploton silver nitrate staining, and spatially resolved lacunar morphology was quantified using high-resolution backscattered scanning electron microscopy (bSEM). ResultsAt the tissue level, reproduction induced distinct skeletal responses, characterized by reversible cortical bone loss and persistent trabecular deterioration. Cortical bone loss during lactation was spatially asymmetric and confined to the posterior cortex. Fluorochrome labeling further resolved surface-specific remodeling patterns during reproduction, including endocortical resorption at the posterior cortex and sustained deposition at anterior endocortical and posterior periosteal surfaces. At the cellular level, osteocyte PLR activity increased during lactation in WT mice and returned after weaning, whereas no changes were observed in cKO mice. Consistently, lacunar size increased during lactation and returned toward baseline after weaning in WT mice but remained unchanged in cKO mice. Spatially resolved analysis demonstrated that lacunar remodeling was heterogeneous across cortical thickness. At the anterior cortex, lacunar enlargement occurred near the endocortical surface during lactation and was reversible after weaning. In contrast, at the posterior cortex, lacunar enlargement occurred near the periosteal surface and persisted after weaning. These spatial patterns corresponded to cortical regions enriched with newly formed osteocytes, whereas pre-existing osteocytes exhibited minimal changes. This spatial heterogeneity was absent in cKO mice. ConclusionOsteocyte PLR-mediated lacunar remodeling during reproduction is spatially heterogeneous and varies with osteocyte location and relative age. These findings demonstrate that recovery at the tissue level does not necessarily extend to the osteocyte microenvironment and identify osteocyte PLR-mediated lacunar remodeling as a spatially structured and potentially persistent component of reproductive skeletal adaptation. Together, these results highlight a previously unrecognized layer of maternal skeletal health.