Heart failure is often accompanied by titin-dependent myocardial stiffness. Phosphorylation of titin by cGMP-dependent protein kinase I (PKGI) increases cardiomyocyte distensibility. The upstream pathways stimulating PKGI-mediated titin phosphorylation are unclear. We studied whether C-type natriuretic peptide (CNP), via its guanylyl cyclase-B (GC-B) receptor and cGMP/PKGI signaling, modulates titin-based ventricular compliance. To dissect GC-B–mediated effects of endogenous CNP in cardiomyocytes, we generated mice with cardiomyocyte-restricted GC-B deletion (CM GC-B–KO mice). The impact on heart morphology and function, myocyte passive tension, and titin isoform expression and phosphorylation was studied at baseline and after increased afterload induced by transverse aortic constriction (TAC). Pressure overload increased left ventricular endothelial CNP expression, with an early peak after 3 days. Concomitantly, titin phosphorylation at Ser4080, the site phosphorylated by PKGI, was augmented. Notably, in CM GC-B–KO mice this titin response was abolished. TAC-induced hypertrophy and fibrosis were not different between genotypes. However, the KO mice presented mild systolic and diastolic dysfunction together with myocyte stiffness, which were not observed in control littermates. In vitro, recombinant PKGI rescued reduced titin-Ser4080 phosphorylation and reverted passive stiffness of GC-B–deficient cardiomyocytes. CNP-induced activation of GC-B/cGMP/PKGI signaling in cardiomyocytes provides a protecting regulatory circuit preventing titin-based myocyte stiffening during early phases of pressure overload.
Konstanze Michel, Melissa Herwig, Franziska Werner, Katarina Špiranec Spes, Marco Abeßer, Kai Schuh, Swati Dabral, Andreas Mügge, Hideo A. Baba, Boris V. Skryabin, Nazha Hamdani, Michaela Kuhn
Many mutation analyses of the HBV genome have been performed in the search for new prognostic markers. However, the Kozak sequence preceding precore was covered only infrequently in these analyses. In this study, the HBV core promoter/precore region was sequenced in serum samples from European inactive HBV carriers. Quadruple mutation GCAC1809-1812TTCT was found with a high prevalence of 42% in the Kozak sequence preceding precore among all HBV genotypes. GCAC1809-1812TTCT was strongly associated with coexistence of basal core promoter (BCP) double mutation A1762T/G1764A and lower HBV DNA levels. In vitro GCAC1809-1812TTCT lead to drastically diminished synthesis of pregenomic RNA (pgRNA), precore mRNA, core, HBsAg, and HBeAg. Calculation of the pgRNA secondary structure suggests a destabilization of the pgRNA structure by A1762T/G1764A that was compensated by GCAC1809-1812TTCT. In 125 patients with HBV-related cirrhosis, GCAC1809-1812TTCT was not detected. While a strong association of GCAC1809-1812TTCT with inactive carrier status was observed, BCP double mutation was strongly correlated with cirrhosis, but this was only observed in absence of GCAC1809-1812TTCT. In conclusion, our data reveal that GCAC1809-1812TTCT is highly prevalent in inactive carriers and acts as a compensatory mutation for BCP double mutation. GCAC1809-1812TTCT seems to be a biomarker of good prognosis in HBV infection.
Kai-Henrik Peiffer, Catrina Spengler, Michael Basic, Bingfu Jiang, Lisa Kuhnhenn, Wiebke Obermann, Tobias Zahn, Mirco Glitscher, Alessandro Loglio, Floriana Facchetti, Gert Carra, Alica Kubesch, Johannes Vermehren, Viola Knop, Christiana Graf, Julia Dietz, Fabian Finkelmeier, Eva Herrmann, Jonel Trebicka, Arnold Grünweller, Stefan Zeuzem, Christoph Sarrazin, Pietro Lampertico, Eberhard Hildt
Intratumoral immune infiltrate was recently reported in gastrointestinal stromal tumors (GISTs). However, the tumor-intrinsic factors that dictate GIST immunogenicity are still largely undefined. To shed light on this issue, a large cohort (82 samples) of primary untreated GISTs, representative of major clinicopathological variables, was investigated by an integrated immunohistochemical, transcriptomic, and computational approach. Our results indicate that tumor genotype, location, and malignant potential concur to shape the immunogenicity of primary naive GISTs. Immune infiltration was greater in overt GISTs compared with that in lesions with limited malignant potential (miniGISTs), in KIT/PDGFRA-mutated tumors compared with that in KIT/PDGFRA WT tumors, and in PDGFRA-mutated compared with KIT-mutated GISTs. Within the KIT-mutated subset, a higher degree of immune colonization was detected in the intestine. Immune hot tumors showed expression patterns compatible with a potentially proficient but curbed antigen-specific immunity, hinting at sensitivity to immunomodulatory treatments. Poorly infiltrated GISTs, primarily KIT/PDGFRA WT intestinal tumors, showed activation of Hedgehog and WNT/β-catenin immune excluding pathways. This finding discloses a potential therapeutic vulnerability, as the targeting of these pathways might prove effective by both inhibiting pro-oncogenic signals and fostering antitumor immune responses. Finally, an intriguing anticorrelation between immune infiltration and ANO1/DOG1 expression was observed, suggesting an immunomodulatory activity for anoctamin-1.
Daniela Gasparotto, Marta Sbaraglia, Sabrina Rossi, Davide Baldazzi, Monica Brenca, Alessia Mondello, Federica Nardi, Dominga Racanelli, Matilde Cacciatore, Angelo Paolo Dei Tos, Roberta Maestro
WNK1 (with no lysine [K] kinase 1) is an atypical kinase protein ubiquitously expressed in humans and mice. A mutation in its encoding gene causes hypertension in humans, which is associated with abnormal ion homeostasis. WNK1 is critical for in vitro decidualization in human endometrial stromal cells, thereby demonstrating its importance in female reproduction. Using a mouse model, WNK1 was ablated in the female reproductive tract to define its in vivo role in uterine biology. Loss of WNK1 altered uterine morphology, causing endometrial epithelial hyperplasia, adenomyotic features, and a delay in embryo implantation, ultimately resulting in compromised fertility. Combining transcriptomic, proteomic, and interactomic analyses revealed a potentially novel regulatory pathway whereby WNK1 represses AKT phosphorylation through protein phosphatase 2A (PP2A) in endometrial cells from both humans and mice. We show that WNK1 interacted with PPP2R1A, the alpha isoform of the PP2A scaffold subunit. This maintained the levels of PP2A subunits and stabilized its activity, which then dephosphorylated AKT. Therefore, loss of WNK1 reduced PP2A activity, causing AKT hypersignaling. Using FOXO1 as a readout of AKT activity, we demonstrate that there was escalated FOXO1 phosphorylation and nuclear exclusion, leading to a disruption in the expression of genes that are crucial for embryo implantation.
Ru-pin Alicia Chi, Tianyuan Wang, Chou-Long Huang, San-pin Wu, Steven L. Young, John P. Lydon, Francesco J. DeMayo
Epigenetic dysregulation is implicated in the pathogenesis of lupus. We performed a longitudinal analysis to assess changes in DNA methylation in lupus neutrophils over 4 years of follow-up and across disease activity levels using 229 patient samples. We demonstrate that DNA methylation profiles in lupus are partly determined by ancestry-associated genetic variations and are highly stable over time. DNA methylation levels in 2 CpG sites correlated significantly with changes in lupus disease activity. Progressive demethylation in SNX18 was observed with increasing disease activity in African American patients. Importantly, demethylation of a CpG site located within GALNT18 was associated with the development of active lupus nephritis. Differentially methylated genes between African American and European American lupus patients include type I IFN–response genes such as IRF7 and IFI44, and genes related to the NF-κB pathway. TREML4, which plays a vital role in TLR signaling, was hypomethylated in African American patients and demonstrated a strong cis–methylation quantitative trait loci (cis-meQTL) effect among 8855 cis-meQTL associations identified in our study.
Patrick Coit, Lourdes Ortiz-Fernandez, Emily E. Lewis, W. Joseph McCune, Kathleen Maksimowicz-McKinnon, Amr H. Sawalha
Inosine triphosphate pyrophosphatase (ITPA) hydrolyzes inosine triphosphate (ITP) and other deaminated purine nucleotides to the corresponding nucleoside monophosphates. In humans, ITPA deficiency causes severe encephalopathy with epileptic seizure, microcephaly, and developmental retardation. In this study, we established neural stem cell–specific Itpa–conditional KO mice (Itpa-cKO mice) to clarify the effects of ITPA deficiency on the neural system. The Itpa-cKO mice showed growth retardation and died within 3 weeks of birth. We did not observe any microcephaly in the Itpa-cKO mice, although the female Itpa-cKO mice did show adrenal hypoplasia. The Itpa-cKO mice showed limb-clasping upon tail suspension and spontaneous and/or audiogenic seizure. Whole-cell patch-clamp recordings from entorhinal cortex neurons in brain slices revealed a depolarized resting membrane potential, increased firing, and frequent spontaneous miniature excitatory postsynaptic current and miniature inhibitory postsynaptic current in the Itpa-cKO mice compared with ITPA-proficient controls. Accumulated ITP or its metabolites, such as cyclic inosine monophosphates, or RNA containing inosines may cause membrane depolarization and hyperexcitability in neurons and induce the phenotype of ITPA-deficient mice, including seizure.
Yuichiro Koga, Daisuke Tsuchimoto, Yoshinori Hayashi, Nona Abolhassani, Yasuto Yoneshima, Kunihiko Sakumi, Hiroshi Nakanishi, Shinya Toyokuni, Yusaku Nakabeppu
Infection-driven inflammation in pregnancy is a major cause of spontaneous preterm birth (PTB). Both systemic infection and bacterial ascension through the vagina/cervix to the amniotic cavity are strongly associated with PTB. However, the contribution of maternal or fetal inflammatory responses in the context of systemic or localized models of infection-driven PTB is not well defined. Here, using intraperitoneal or intraamniotic LPS challenge, we examined the necessity and sufficiency of maternal and fetal Toll-like receptor (TLR) 4 signaling in induction of inflammatory vigor and PTB. Both systemic and local LPS challenge promoted induction of inflammatory pathways in uteroplacental tissues and induced PTB. Restriction of TLR4 expression to the maternal compartment was sufficient for induction of LPS-driven PTB in either systemic or intraamniotic challenge models. In contrast, restriction of TLR4 expression to the fetal compartment failed to induce LPS-driven PTB. Vav1-Cre–mediated genetic deletion of TLR4 suggested a critical role for maternal immune cells in inflammation-driven PTB. Further, passive transfer of WT in vitro–derived macrophages and dendritic cells to TLR4-null gravid females was sufficient to induce an inflammatory response and drive PTB. Cumulatively, these findings highlight the critical role for maternal regulation of inflammatory cues in induction of inflammation-driven parturition.
Monica Cappelletti, Jessica R. Doll, Traci E. Stankiewicz, Matthew J. Lawson, Vivien Sauer, Bingqiang Wen, Vladimir V. Kalinichenko, Xiaofei Sun, Tamara Tilburgs, Senad Divanovic
Adult liver has enormous regenerative capacity; it can regenerate after losing two-thirds of its mass while sustaining essential metabolic functions. How the liver balances dual demands for increased proliferative activity with maintenance of organ function is unknown but essential to prevent liver failure. Using partial hepatectomy (PHx) in mice to model liver regeneration, we integrated single-cell RNA- and ATAC-Seq to map state transitions in approximately 13,000 hepatocytes at single-cell resolution as livers regenerated, and validated key findings with IHC, to uncover how the organ regenerates hepatocytes while simultaneously fulfilling its vital tissue-specific functions. After PHx, hepatocytes rapidly and transiently diversified into multiple distinct populations with distinct functional bifurcation: some retained the chromatin landscapes and transcriptomes of hepatocytes in undamaged adult livers, whereas others transitioned to acquire chromatin landscapes and transcriptomes of fetal hepatocytes. Injury-related signaling pathways known to be critical for regeneration were activated in transitioning hepatocytes, and the most fetal-like hepatocytes exhibited chromatin landscapes that were enriched with transcription factors regulated by those pathways.
Tianyi Chen, Sehhoon Oh, Simon Gregory, Xiling Shen, Anna Mae Diehl
The nonimmune roles of Tregs have been described in various tissues, including the BM. In this study, we comprehensively phenotyped marrow Tregs, elucidating their key features and tissue-specific functions. We show that marrow Tregs are migratory and home back to the marrow. For trafficking, marrow Tregs use S1P gradients, and disruption of this axis allows for specific targeting of the marrow Treg pool. Following Treg depletion, the function and phenotype of both mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) was impaired. Transplantation also revealed that a Treg-depleted niche has a reduced capacity to support hematopoiesis. Finally, we found that marrow Tregs are high producers of IL-10 and that Treg-secreted IL-10 has direct effects on MSC function. This is the first report to our knowledge revealing that Treg-secreted IL-10 is necessary for stromal cell maintenance, and our work outlines an alternative mechanism by which this cytokine regulates hematopoiesis.
Virginia Camacho, Victoria R. Matkins, Sweta B. Patel, Jeremie M. Lever, Zhengqin Yang, Li Ying, Ashley E. Landuyt, Emma C. Dean, James F. George, Henry Yang, Paul Brent Ferrell, Craig L. Maynard, Casey T. Weaver, Heth R. Turnquist, Robert S. Welner
Successful implantation is associated with a unique spatial pattern of vascular remodeling, characterized by profound peripheral neovascularization surrounding a periembryo avascular niche. We hypothesized that hyaluronan controls the formation of this distinctive vascular pattern encompassing the embryo. This hypothesis was evaluated by genetic modification of hyaluronan metabolism, specifically targeted to embryonic trophoblast cells. The outcome of altered hyaluronan deposition on uterine vascular remodeling and postimplantation development were analyzed by MRI, detailed histological examinations, and RNA sequencing of uterine NK cells. Our experiments revealed that disruption of hyaluronan synthesis, as well as its increased cleavage at the embryonic niche, impaired implantation by induction of decidual vascular permeability, defective vascular sinus folds formation, breach of the maternal-embryo barrier, elevated MMP-9 expression, and interrupted uterine NK cell recruitment and function. Conversely, enhanced deposition of hyaluronan resulted in the expansion of the maternal-embryo barrier and increased diffusion distance, leading to compromised implantation. The deposition of hyaluronan at the embryonic niche is regulated by progesterone-progesterone receptor signaling. These results demonstrate a pivotal role for hyaluronan in successful pregnancy by fine-tuning the periembryo avascular niche and maternal vascular morphogenesis.
Ron Hadas, Eran Gershon, Aviad Cohen, Ofir Atrakchi, Shlomi Lazar, Ofra Golani, Bareket Dassa, Michal Elbaz, Gadi Cohen, Raya Eilam, Nava Dekel, Michal Neeman
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