Cell biology
Abstract
Laminin-α2-related Congenital Muscular Dystrophy (LAMA2-CMD) is a severe neuromuscular disorder caused by mutations in the LAMA2 gene, leading to loss of heterotrimers laminin-211/221, key components of the skeletal muscle extracellular matrix. Their absence disrupts adhesion between the cytoskeleton and extracellular matrix, resulting in progressive muscle wasting. Laminin-211/221 interacts with adhesion complexes such as the dystrophin/Utrophin glycoprotein complex and the α7β1-integrin. However, the regulatory mechanisms of these laminin-binding complexes and the broader role of laminin’s influence on the formation of the macromolecular network in skeletal muscle remain unclear. We previously demonstrated that mouse laminin-111 delivered in the dyW⁻/⁻ mouse model of LAMA2-CMD prevented disease progression, improved strength, and extended survival. We hypothesize that laminin-111, the embryonic laminin isoform, restores key adhesion-signaling networks. Using spatial-proteomics on patient and mouse muscle, we identified loss of essential signaling components: heat shock proteins 27 and 70, c-Jun N-terminal kinase, and glucose transporter 1 in laminin-α2 deficient muscle. Treatment with recombinant human laminin-111 (rhLAM-111) restored protein localization, reduced ROS, and promoted glycolytic, pro-survival signaling. These findings highlight laminin’s role in maintaining muscle homeostasis and metabolism and support the therapeutic potential of rhLAM-111 for treating LAMA2-CMD by restoring adhesion and intracellular signaling in dystrophic muscle.
Authors
Hailey J. Hermann, Ryan D. Wuebbles, Marisela Dagda, Axel Munoz, Lauren L. Parker, Paula C. C Guzman, Lola T. Byrne, Steven A. Moore, Dean J. Burkin
Abstract
Impaired wound healing poses a major and increasingly frequent health problem. Among the key players in the healing process are fibroblasts, but their metabolic profile in healing wounds is largely unknown. Using a combination of transcriptomics, targeted proteomics and metabolomics, we identified retinol metabolism as a top regulated pathway in wound fibroblasts. This is functionally relevant, since even a mild retinol deficiency caused a delay in wound closure and re-epithelialization, which mainly resulted from misdirected keratinocyte migration on the new granulation tissue. Quantitative proteomics identified integrin alpha 11 (Itga11) as a less abundant protein in wounds of mice subjected to a retinol-deficient diet. Reduced levels of this fibroblast-specific protein likely altered the granulation tissue matrix, which in turn affected re-epithelialization. These results provide a comprehensive overview on the transcriptome, proteome and metabolome of wound fibroblasts and identify retinol metabolism in fibroblasts as a key regulator of tissue repair.
Authors
Till Wüstemann, Elizabeta Madzharova, Mateusz S. Wietecha, Norbert B. Ghyselinck, Marcus Höring, Gerhard Liebisch, Nicola Zamboni, Ulrich auf dem Keller, Sabine Werner
Abstract
IDH1/2 mutations (IDHmut) increase methylation of DNA and histones in gliomas. IDHmut inhibitors are effective against low-grade IDHmut gliomas, but new strategies against high grade IDHmut gliomas are needed. Although histone deacetylase inhibitors (HDACi) are ineffective against IDHwt glioblastoma (GBM), their potential in IDHmut gliomas has not been extensively studied. We previously established that IDHmut gliomas are more sensitive to HDACi than IDHwt GBM. Here we show that IDHmut is associated with greater sensitivity to HDACi only in glioma, not in IDHmut chondrosarcoma or cholangiocarcinoma. While HDACi induced more histone acetylation and gene regulation in IDHmut glioma than in IDHwt GBM, such acetylation was mostly within gene deserts, whereas IDHmut glioma promoters paradoxically lost histone acetylation. Two mediators of HDACi resistance, YAP and TAZ, were methylated and suppressed in IDHmut gliomas, but not in other IDHmut cancers. Inducing YAP or TAZ expression in IDHmut gliomas conferred resistance to HDACi. Finally, belinostat extended in vivo survival only in IDHmut glioma models, not in IDHmut GBM models. Our findings provide a mechanistic rationale for further studies of HDACi in IDHmut glioma patients, as well as the potential use of YAP/TAZ as a biomarker of HDACi sensitivity in cancers.
Authors
Thomas K. Sears, Matthew McCord, Wenxia Wang, Alicia Steffens, Kathleen McCortney, Rahul Chaliparambil, Jann N. Sarkaria, Craig M. Horbinski
Abstract
Coronary artery disease (CAD) is the leading cause of mortality worldwide, with macrophages playing a central role in shaping the inflammatory environment through cytokines, chemokines, and other mediators. Long noncoding RNAs (lncRNAs) are emerging as key regulators of cellular processes due to their interactions with DNA, RNA, microRNAs, and proteins, positioning them as promising therapeutic targets. Through integrative transcriptomic analysis, we identified SPANXA2-OT1 as a primate-specific lncRNA with a potential role in macrophage-mediated inflammation in CAD. Functional studies in primary human macrophages demonstrated that SPANXA2-OT1 is induced by inflammatory stimulation, localized to the cytoplasm, and exerts regulatory effects on chemokine expression and macrophage chemotaxis. Mechanistically, SPANXA2-OT1 acts as a molecular sponge for microRNA-338, thereby influencing the expression of interleukin-8 (IL-8), a critical mediator of monocyte recruitment and inflammatory signaling. Collectively, these findings establish SPANXA2-OT1 as a human-specific regulator of inflammatory pathways in CAD and highlight its translational potential as both a biomarker and therapeutic target.
Authors
Prabhash Kumar Jha, Sarvesh Chelvanambi, Yuto Nakamura, Lucas Yuji Umesaki Itto, Aatira Vijay, Adrien Lupieri, Miguel Cantadori Barbeiro, Thanh-Dat Le, Caio Borges Nascimento, Taku Kasai, Mary C. Whelan, Daiki Hosokawa, Dakota Becker-Greene, Sasha A. Singh, Elena Aikawa, Shizuka Uchida, Masanori Aikawa
Abstract
The unfolded protein response (UPR), triggered by endoplasmic reticulum (ER) stress, comprises distinct pathways orchestrated by conserved molecular sensors. Although several of these components have been suggested to protect cardiomyocytes from ischemic injury, their precise functions and mechanisms remain elusive. In this study, we observed a marked increase in glucose-regulated protein 94 (GRP94) expression at the border zone of cardiac infarct in a mouse model. GRP94 overexpression ameliorated post-infarction myocardial damage and reduced infarct size. Conversely, GRP94 deficiency exacerbated myocardial dysfunction and infarct size. Mechanistically, GRP94 alleviated hypoxia-induced mitochondrial fragmentation, whereas its depletion exacerbated this fragmentation. Molecular investigations revealed that GRP94 specifically facilitated the cleavage of Opa1 into L-Opa1, but not S-Opa1. The study further elucidated that under hypoxic conditions, the binding shift of Yy1 from lncRNA Oip5os1 to AI662270 promoted Yy1’s binding on the GRP94 promoter, thereby enhancing GRP94 expression. AI662270 attenuated mitochondrial over-fragmentation and ischemic injury after myocardial infarction similarly to GRP94. Moreover, coimmunoprecipitation coupled with LC-MS/MS identified the interaction of GRP94 with Anxa2, which regulates Akt1 signaling to maintain L-Opa1 levels. Overall, these findings unveiled what we believe is a novel role for the AI662270/GRP94 axis in linking ER stress to mitochondrial dynamics regulation, proposing new therapeutic avenues for managing cardiovascular conditions through ER stress modulation.
Authors
Suling Ding, Wen Liu, Zhiwei Zhang, Xiyang Yang, Dili Sun, Jianfu Zhu, Xiaowei Zhu, Shijun Wang, Mengshi Xie, Hongyu Shi, Junbo Ge, Xiangdong Yang
Abstract
Intestinal epithelial barrier-integrity is essential for human health, and its disruption induces and exacerbates intestinal inflammatory disorders. While the epithelial cytoskeleton is critical for maintaining gut barrier-integrity, the role of septins- a family of GTP-binding, cytoskeletal proteins- is largely unknown. This highlights an important knowledge gap as dysfunction of septins, and specifically septin 9 (SEPT9), is associated with intestinal pathologies. We determined that SEPT9 localizes to the apical junctions of intestinal epithelial cells (IECs), overlapping with both tight and adherens junctions. IEC-specific ablation of SEPT9 in mice resulted in leaky gut, due to mislocalization of junctional proteins, and increased susceptibility to experimental colitis. Consistently, SEPT9 expression was significantly reduced in intestinal mucosa of inflammatory bowel disease (IBD) patients. Using affinity-purification mass spectrometry, super-resolution imaging, and genetic knockout, we determined that SEPT9 interacts with and is necessary to recruit non-muscle myosin IIC (NMIIC) to the IEC peri-junctional actomyosin belt. Loss of NMIIC also caused IEC barrier disruption. In summary, SEPT9 regulates intestinal barrier-integrity by supporting the assembly of tight and adherens junctions through NMIIC recruitment to the actomyosin belt. The septin cytoskeleton safeguards the intestinal mucosa during acute inflammation, and its disruption in IBD suggests a loss of this protective function.
Authors
Nayden G. Naydenov, Gaizun Hu, Dominik Robak, Atif Zafar, Khosiyat Makhmudova, Susana Lechuga, Yuta Ohno, Naseer Sangwan, Saikat Bandyopadhyay, Ryan Musich, Erin Jeffery, Lei Sun, Armando Marino-Melendez, Florian Rieder, Gloria Sheynkman, Andrei I. Ivanov, Seham Ebrahim
Abstract
Impairment of desmosomal cell-cell adhesion leads to life-threatening diseases such as the autoimmune skin blistering disorder pemphigus vulgaris (PV). Disease management strategies that stabilize intercellular adhesion, in addition to the existing immunosuppression therapies, may result in improved clinical outcomes. Previous findings showed that the serine protease inhibitor SERPINB5 promotes intercellular adhesion by binding to and regulating the localization of the desmosomal adapter molecule desmoplakin (DSP) at the plasma membrane. We here show that SERPINB5 overexpression prevents PV-IgG-mediated loss of cell-cell adhesion and DSP dissociation from the cell membrane. We mechanistically demonstrate that SERPINB5 loss deregulates TGF-β signalling, a pathway known to destabilize DSP in keratinocytes. TGF-β signalling was also activated in skin biopsies of PV patients and keratinocytes treated with PV autoantibodies, suggesting a contribution to disease. Inhibition of TGF-β signaling ameliorated PV-IgG-mediated loss of cell-cell adhesion, increased DSP membrane expression, and prevented PV-IgG-induced blister formation in a human ex-vivo skin model. Together, SERPINB5 modulates DSP and intercellular adhesion through the regulation of TGF-β signalling. Further, TGF-β signalling was identified as a potential target for pemphigus treatment.
Authors
Maitreyi Rathod, Mariam Petrosyan, Aude Zimmermann, Maike Märker, Tobias Gosau, Henriette Franz, Tomás Cunha, Dario Didona, Michael Hertl, Enno Schmidt, Volker Spindler
Abstract
Rheumatoid arthritis (RA) is a common systemic autoimmune disorder. Fibroblast-like synoviocytes (FLS) have emerged as an attractive target for non-immunosuppressive RA therapy, but there are no approved drugs targeting FLS. The receptor protein tyrosine phosphatase sigma (PTPRS) negatively regulates FLS migration and has been proposed as a target for FLS-directed RA therapy. Here we examined the impact of sequence variations on efficacy of an FLS-targeted biologic comprised of Fc-fused PTPRS immunoglobulin (IgG)-like domains Ig1 and Ig2 (Ig1&2-Fc). Engineering the linker and Fc tag improved effectiveness of human Ig1&2-Fc in assays of FLS migration and a mouse model of arthritis. Treatment of mice with Ig1&2-Fc over four months revealed no signs of toxicity or organ pathology. Finally, we show potential of Ig1&2-Fc co-administration in combination or as a bispecific fusion with a tumor necrosis factor alpha inhibitor (TNFi). Combination treatment of mouse tumor necrosis factor receptor 2 (mTnfr2) with Ig1&2-Fc resulted in increased efficacy in suppressing arthritis beyond single agent treatment. When administered as a dual-action bispecific, Ig1&2 fused to mTnfr2 proved more efficacious at suppressing arthritis than mTnfr2 alone. This study illustrates the potential of Ig1&2-Fc as a combination or bispecific therapy with DMARDs to improve patient outcomes in RA.
Authors
Sterling H. Ramsey, Zixuan Zhao, Megan C. Lee, Thales Hein da Rosa, Ava C. Schneider, Miriam Bollmann, Nour Dada, Katie Frizzi, May M. Han, Jaeyeon Kim, Martina Zoccheddu, Nigel A. Calcutt, Gary S. Firestein, James W. Bryson, Mattias N.D. Svensson, Eugenio Santelli, Stephanie M. Stanford, Nunzio Bottini
Abstract
Cranial neural crest cells (CNCs) play a critical role in craniofacial bone morphogenesis, engaging in intricate interactions with various molecular signals to ensure proper development, yet the molecular scaffolds coordinating these processes remain incompletely defined. Here, we identify neurofibromin 2 (Nf2) as a critical regulator to direct CNC-derived skull morphogenesis. Genetic ablation of Nf2 in murine CNCs causes severe craniofacial anomalies, featuring declined proliferation and increased apoptosis in osteoprogenitors, impaired type I collagen biosynthesis and trafficking, and aberrant osteogenic mineralization. Mechanistically, we uncover that Nf2 serves as a molecular linker that individually interacts with FGF receptor 1 (FGFR1) and Akt through spatially segregated phosphor-sites, and structural modeling and mutagenesis identified Ser10 and Thr230 as essential residues, with Thr230 mutation selectively ablating Akt binding while preserving FGFR1 association. Strikingly, Akt inhibition phenocopied Nf2 deficiency, reducing collagen production and Nf2 phosphorylation, whereas phospho-mimetic Nf2 (T230D) rescued CNC-derived osteogenic defects in Nf2-mutant animals. Our findings underscore the physiological significance of Nf2 as a phosphorylation-operated scaffold licensing the FGFR1/AKT axis to regulate collagen type I biogenesis and trafficking, ensuring normal CNC-derived osteogenesis and craniofacial bone development, thus exposing the Nf2/FGFR1/AKT signaling axis as a therapeutic target and promising advancements in treatment of craniofacial anomalies.
Authors
Yuping Huang, Junguang Liao, Panpan Shen, Yiliang He, Fuju Sun, Qi Zhang, Changlin Zheng, Xingen Zhang, Haibo Li, Guiqian Chen
Abstract
Recent experimental and epidemiologic data have strongly associated air pollution in the pathogenesis of insulin resistance and type 2 diabetes mellitus. We explored the effect of inhalational exposure to concentrated ambient particulate matter smaller than 2.5 μm (PM2.5), or filtered air, using a whole-body inhalation system (6 hours/day, 5 days/week) for 24 weeks on metabolism and brown adipose tissue (BAT) function. Mechanistic evaluation of insulin resistance, glucose uptake with 18F-fluorodeoxyglucose positron emission tomography, alongside evaluation for differentially methylated regions, chromatin accessibility, and differential expression of genes was performed. PM2.5 exposure impaired metabolism through changes in key BAT transcriptional programs involved in redox stress, lipid deposition, fibrosis, and altered thermogenesis. Significant differential methylation and widespread chromatin remodeling was noted in BAT with PM2.5. Integrated analysis uncovered a role for the histone deacetylase HDAC9 and histone demethylase KDM2B. The latter demethylates Lys-4 and Lys-36 of histone H3. Specifically, studies using ChIP combined with quantitative PCR confirmed HDAC9 and KDM2B occupancy and reduced H3K36me2 on the promoter of target BAT genes in PM2.5 mice, while Hdac9/Kdm2b knockdown and overexpression increased and reduced BAT metabolism, respectively. Collectively, our results provide insights into air pollution exposure and changes in BAT and metabolism.
Authors
Rengasamy Palanivel, Jean-Eudes Dazard, Bongsoo Park, Sarah Costantino, Skanda T. Moorthy, Armando Vergara-Martel, Elaine Ann Cara, Jonnelle Edwards-Glenn, Shyam Biswal, Lung Chi Chen, Mukesh K. Jain, Francesco Paneni, Sanjay Rajagopalan
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