Browsing by Subject "CELL"

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  • Hurler, Julia; Sorensen, Karen K.; Fallarero, Adyary; Vuorela, Pia; Skalko-Basnet, Natasa (2013)
  • Heybrock, Saskia; Kanerva, Kristiina; Meng, Ying; Ing, Chris; Liang, Anna; Xiong, Zi-Jian; Weng, Xialian; Kim, Young Ah; Collins, Richard; Trimble, William; Pomes, Regis; Prive, Gilbert G.; Annaert, Wim; Schwake, Michael; Heeren, Joerg; Lullmann-Rauch, Renate; Grinstein, Sergio; Ikonen, Elina; Saftig, Paul; Neculai, Dante (2019)
    The intracellular transport of cholesterol is subject to tight regulation. The structure of the lysosomal integral membrane protein type 2 (LIMP-2, also known as SCARB2) reveals a large cavity that traverses the molecule and resembles the cavity in SR-B1 that mediates lipid transfer. The detection of cholesterol within the LIMP-2 structure and the formation of cholesterol - like inclusions in LIMP-2 knockout mice suggested the possibility that LIMP2 transports cholesterol in lysosomes. We present results of molecular modeling, crosslinking studies, microscale thermophoresis and cell-based assays that support a role of LIMP-2 in cholesterol transport. We show that the cavity in the luminal domain of LIMP-2 can bind and deliver exogenous cholesterol to the lysosomal membrane and later to lipid droplets. Depletion of LIMP-2 alters SREBP-2-mediated cholesterol regulation, as well as LDL-receptor levels. Our data indicate that LIMP-2 operates in parallel with Niemann Pick (NPC)-proteins, mediating a slower mode of lysosomal cholesterol export.
  • Hämetoja, Hanna; Mäkitie, Antti; Bäck, Leif; Leivo, Ilmo; Haglund, Caj; Sorsa, Timo; Hagström, Jaana (2021)
    Knowledge on the role of matrix metalloproteinases (MMPs) in adenoid cystic carcinoma (ACC) is limited. MMPs are capable of degrading almost all extracellular and pericellular components to promote invasion and metastasis. This study aimed to evaluate the immunohistochemical expression of MMP-7,-8,-9,-15, and-25 in ACC and to relate the results with clinicopathological factors and survival. The study included 68 patients with minor salivary gland ACC treated at the Helsinki University Hospital (Helsinki, Finland) in 1974-2012. Samples from 52 patients were available, consisting of 44 primary tumours and eight recurrent tumours. We scored immunostaining of MMP-7,-8,-9,-15, and-25 and analysed the immunoscore against clinical and pathological parameters using statistical correlation test. MMP-9 immunoexpression in pseudocysts of ACC and in peritumoural inflammatory cells associated with better survival and fewer treatment failures. High tumoural MMP-7 and-25 associated with better survival. High tumoural MMP-15 associated with poorer survival and high tumoural MMP-9 with advanced stage and regional recurrences. Tumour cells did not show MMP-8 immunopositivity. These results suggest that MMP-9 may contribute to ACC carcinogenesis in different roles. MMP-7,-8, and-9 can stimulate signalling pathways that may promote tissue modulation and metastatic potential. MMP-15 and-25 may reflect prognosis.
  • Santinho, Alexandre; Salo, Veijo T.; Chorlay, Aymeric; Li, Shiqian; Zhou, Xin; Omrane, Mohyeddine; Ikonen, Elina; Thiam, Abdou Rachid (2020)
    Lipid droplet (LD) biogenesis begins in the endoplasmic reticulum (ER) bilayer, but how the ER topology impacts this process is unclear. An early step in LD formation is nucleation, wherein free neutral lipids, mainly triacylglycerols (TGs) and sterol esters (SEs), condense into a nascent LD. How this transition occurs is poorly known. Here, we found that LDs preferably assemble at ER tubules, with higher curvature than ER sheets, independently of the LD assembly protein seipin. Indeed, the critical TG concentration required for initiating LD assembly is lower at curved versus flat membrane regions. In agreement with this finding, flat ER regions bear higher amounts of free TGs than tubular ones and present less LDs. By using an in vitro approach, we discovered that the presence of free TGs in tubules is energetically unfavorable, leading to outflow of TGs to flat membrane regions or condensation into LDs. Accordingly, in vitro LD nucleation can be achieved by the sole increase of membrane curvature. In contrast to TGs, the presence of free SEs is favored at tubules and increasing SE levels is inhibitory to the curvature-induced nucleation of TG LDs. Finally, we found that seipin is enriched at ER tubules and controls the condensation process, preventing excessive tubule-induced nucleation. The absence of seipin provokes erratic LD nucleation events determined by the abundance of ER tubules. In summary, our data indicate that membrane curvature catalyzes LD assembly.
  • Lehtonen, Sanna (2020)
    Metformin is the most commonly prescribed drug for treating type 2 diabetes mellitus (T2D). Its mechanisms of action have been under extensive investigation, revealing that it has multiple cellular targets, either direct or indirect ones, via which it regulates numerous cellular pathways. Diabetic kidney disease (DKD), the serious complication of T2D, develops in up to 50% of the individuals with T2D. Various mechanisms contribute to the development of DKD, including hyperglycaemia, dyslipidemia, oxidative stress, chronic low-grade inflammation, altered autophagic activity and insulin resistance, among others. Metformin has been shown to affect these pathways, and thus, it could slow down or prevent the progression of DKD. Despite several animal studies demonstrating the renoprotective effects of metformin, there is no concrete evidence in clinical settings. This review summarizes the renoprotective effects of metformin in experimental settings. Special emphasis is on the effects of metformin on podocytes, the glomerular epithelial cells that are central in maintaining the glomerular ultrafiltration function.
  • Morello, Francesca; Borshagovski, Daniel; Survila, Mantas; Tikker, Laura; Sadik-Ogli, Samir; Kirjavainen, Anna; Estartús, Nuri; Knaapi, Laura; Lahti, Laura; Törönen, Petri; Mazutis, Linas; Delogu, Alessio; Salminen, Marjo; Achim, Kaia; Partanen, Juha (2020)
    Tegmental nuclei in the ventral midbrain and anterior hindbrain control motivated behavior, mood, memory, and movement. These nuclei contain inhibitory GABAergic and excitatory glutamatergic neurons, whose molecular diversity and development remain largely unraveled. Many tegmental neurons originate in the embryonic ventral rhombomere 1 (r1), where GABAergic fate is regulated by the transcription factor (TF) Tal1. We used single-cell mRNA sequencing of the mouse ventral r1 to characterize the Tal1-dependent and independent neuronal precursors. We describe gene expression dynamics during bifurcation of the GABAergic and glutamatergic lineages and show how active Notch signaling promotes GABAergic fate selection in postmitotic precursors. We identify GABAergic precursor subtypes that give rise to distinct tegmental nuclei and demonstrate that Sox14 and Zfpm2, two TFs downstream of Tal1, are necessary for the differentiation of specific tegmental GABAergic neurons. Our results provide a framework for understanding the development of cellular diversity in the tegmental nuclei.
  • Ajdary, Rubina; Zanjanizadeh Ezazi, Nazanin; Rebelo Correia, Alexandra Maria; Kemell, Marianna; Huan, Siqi; Ruskoaho, Heikki; Hirvonen, Jouni; Santos, Hélder A.; Rojas, Orlando J. (2020)
    A biomaterial system incorporating nanocellulose, poly(glycerol sebacate), and polypyrrole is introduced for the treatment of myocardial infarction. Direct ink writing of the multicomponent aqueous suspensions allows multifunctional lattice structures that not only feature elasticity and electrical conductivity but enable cell growth. They are proposed as cardiac patches given their biocompatibility with H9c2 cardiomyoblasts, which attach extensively at the microstructural level, and induce their proliferation for 28 days. Two model drugs (3i‐1000 and curcumin) are investigated for their integration in the patches, either by loading in the precursor suspension used for extrusion or by direct impregnation of the as‐obtained, dry lattice. In studies of drug release conducted for five months, a slow in vitro degradation of the cardiac patches is observed, which prevents drug burst release and indicates their suitability for long‐term therapy. The combination of biocompatibility, biodegradability, mechanical strength, flexibility, and electrical conductivity fulfills the requirement of the highly dynamic and functional electroresponsive cardiac tissue. Overall, the proposed cardiac patches are viable alternatives for the regeneration of myocardium after infarction through the effective integration of cardiac cells with the biomaterial.
  • Jasmin, Melissa; Ahn, Eun Hee; Voutilainen, Merja H.; Fombonne, Joanna; Guix, Catherine; Viljakainen, Tuulikki; Kang, Seong Su; Yu, Li-ying; Saarma, Mart; Mehlen, Patrick; Ye, Keqiang (2021)
    The netrin-1/DCC ligand/receptor pair has key roles in central nervous system (CNS) development, mediating axonal, and neuronal navigation. Although expression of netrin-1 and DCC is maintained in the adult brain, little is known about their role in mature neurons. Notably, netrin-1 is highly expressed in the adult substantia nigra, leading us to investigate a role of the netrin-1/DCC pair in adult nigral neuron fate. Here, we show that silencing netrin-1 in the adult substantia nigra of mice induces DCC cleavage and a significant loss of dopamine neurons, resulting in motor deficits. Because loss of adult dopamine neurons and motor impairments are features of Parkinson's disease (PD), we studied the potential impact of netrin-1 in different animal models of PD. We demonstrate that both overexpression of netrin-1 and brain administration of recombinant netrin-1 are neuroprotective and neurorestorative in mouse and rat models of PD. Of interest, we observed that netrin-1 levels are significantly reduced in PD patient brain samples. These results highlight the key role of netrin-1 in adult dopamine neuron fate, and the therapeutic potential of targeting netrin-1 signaling in PD.
  • Redchuk, Taras A; Karasev, Maksim M; Verkhusha, Polina V; Donnelly, Sara K; Hülsemann, Maren; Virtanen, Jori; Moore, Henna M; Vartiainen, Maria K; Hodgson, Louis; Verkhusha, Vladislav V (2020)
    Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets. Optogenetic approaches to control protein-protein interactions usually require overexpression of the target proteins. Here the authors integrate intrabodies into near-infrared- and blue-light activatable optogenetic tools to control endogenous proteins in mammalian cells.
  • Nugroho, Robertus Wahyu N.; Harjumäki, Riina; Zhang, Xue; Lou, Yan-Ru; Yliperttula, Marjo; Valle-Delgado, Juan Jose; Österberg, Monika (2019)
    Biomaterials of different nature have been and are widely studied for various biomedical applications. In many cases, biomaterial assemblies are designed to mimic biological systems. Although biomaterials have been thoroughly characterized in many aspects, not much quantitative information on the molecular level interactions between different biomaterials is available. That information is very important, on the one hand, to understand the properties of biological systems and, on the other hand, to develop new composite biomaterials for special applications. This work presents a systematic, quantitative analysis of self- and cross-interactions between films of collagen I (Col I), collagen IV (Col IV), laminin (LN-521), and cellulose nanofibrils (CNF), that is, biomaterials of different nature and structure that either exist in biological systems (e.g., extracellular matrices) or have shown potential for 3D cell culture and tissue engineering. Direct surface forces and adhesion between biomaterials-coated spherical micro-particles and flat substrates were measured in phosphate-buffered saline using an atomic force microscope and the colloidal probe technique. Different methods (Langmuir-Schaefer deposition, spin-coating, or adsorption) were applied to completely coat the flat substrates and the spherical micro particles with homogeneous biomaterial films. The adhesion between biomaterials films increased with the time that the films were kept in contact. The strongest adhesion was observed between Col IV films, and between Col IV and LN-521 films after 30 s contact time. In contrast, low adhesion was measured between CNF films, as well as between CNF and LN-521 films. Nevertheless, a good adhesion between CNF and collagen films (especially Col I) was observed. These results increase our understanding of the structure of biological systems and can support the design of new matrices or scaffolds where different biomaterials are combined for diverse biological or medical applications.
  • Alkasalias, Twana; Alexeyenko, Andrey; Hennig, Katharina; Danielsson, Frida; Lebbink, Robert Jan; Fielden, Matthew; Turunen, S. Pauliina; Lehti, Kaisa; Kashuba, Vladimir; Madapura, Harsha S.; Bozoky, Benedek; Lundberg, Emma; Balland, Martial; Guven, Hayrettin; Klein, George; Gad, Annica K. B.; Pavlova, Tatiana (2017)
    Fibroblasts are a main player in the tumor-inhibitory microenvironment. Upon tumor initiation and progression, fibroblasts can lose their tumor-inhibitory capacity and promote tumor growth. The molecular mechanisms that underlie this switch have not been defined completely. Previously, we identified four proteins over-expressed in cancer-associated fibroblasts and linked to Rho GTPase signaling. Here, we show that knocking out the Ras homolog family member A (RhoA) gene in normal fibroblasts decreased their tumor-inhibitory capacity, as judged by neighbor suppression in vitro and accompanied by promotion of tumor growth in vivo. This also induced PC3 cancer cell motility and increased colony size in 2D cultures. RhoA knockout in fibroblasts induced vimentin intermediate filament reorganization, accompanied by reduced contractile force and increased stiffness of cells. There was also loss of wide F-actin stress fibers and large focal adhesions. In addition, we observed a significant loss of a-smooth muscle actin, which indicates a difference between RhoA knockout fibroblasts and classic cancer-associated fibroblasts. In 3D collagen matrix, RhoA knockout reduced fibroblast branching and meshwork formation and resulted in more compactly clustered tumor-cell colonies in coculture with PC3 cells, which might boost tumor stem-like properties. Coculturing RhoA knockout fibroblasts and PC3 cells induced expression of proinflammatory genes in both. Inflammatory mediators may induce tumor cell stemness. Network enrichment analysis of transcriptomic changes, however, revealed that the Rho signaling pathway per se was significantly triggered only after coculturing with tumor cells. Taken together, our findings in vivo and in vitro indicate that Rho signaling governs the inhibitory effects by fibroblasts on tumor-cell growth.
  • Wasik, Anita A.; Dash, Surjya N.; Lehtonen, Sanna (2019)
    Septins are a conserved family of GTP-binding proteins that assemble into cytoskeletal filaments to function in a highly sophisticated and physiologically regulated manner. Originally septins were discovered in the budding yeast as membrane-associated filaments that affect cell polarity and cytokinesis. In the last decades, much progress has been made in understanding the biochemical properties and cell biological functions of septins. In line with this, mammalian septins have been shown to be involved in various cellular processes, including regulation of cell polarity, cytoskeletal organization, vesicle trafficking, ciliogenesis, and cell-pathogen interactions. A growing number of studies have shown that septins play important roles in tissue and organ development and physiology; yet, little is known about their role in the kidney. In the following review, we discuss the structure and functions of septins in general and summarize the evidence for their presence and roles in the kidney.
  • Selberg, Simona; Yu, Li-Ying; Bondarenko, Olesja; Kankuri, Esko; Seli, Neinar; Kovaleva, Vera; Herodes, Koit; Saarma, Mart; Karelson, Mati (2021)
    The fat mass and obesity-associated protein (FTO), an RNA N-6-methyladenosine (m(6)A) demethylase, is an important regulator of central nervous system development, neuronal signaling and disease. We present here the target-tailored development and biological characterization of small-molecule inhibitors of FTO. The active compounds were identified using high-throughput molecular docking and molecular dynamics screening of the ZINC compound library. In FTO binding and activity-inhibition assays the two best inhibitors demonstrated K-d = 185 nM; IC50 = 1.46 mu M (compound 2) and K-d = 337 nM; IC50 = 28.9 mu M (compound 3). Importantly, the treatment of mouse midbrain dopaminergic neurons with the compounds promoted cellular survival and rescued them from growth factor deprivation induced apoptosis already at nanomolar concentrations. Moreover, both the best inhibitors demonstrated good blood-brain-barrier penetration in the model system, 31.7% and 30.8%, respectively. The FTO inhibitors demonstrated increased potency as compared to our recently developed ALKBH5 m(6)A demethylase inhibitors in protecting dopamine neurons. Inhibition of m(6)A RNA demethylation by small-molecule drugs, as presented here, has therapeutic potential and provides tools for the identification of disease-modifying m(6)A RNAs in neurogenesis and neuroregeneration. Further refinement of the lead compounds identified in this study can also lead to unprecedented breakthroughs in the treatment of neurodegenerative diseases.
  • Piccinini, Filippo; Balassa, Tamas; Carbonaro, Antonella; Diosdi, Akos; Toth, Timea; Moshkov, Nikita; Tasnadi, Ervin A.; Horvath, Peter (2020)
    Today, we are fully immersed into the era of 3D biology. It has been extensively demonstrated that 3D models: (a) better mimic the physiology of human tissues; (b) can effectively replace animal models; (c) often provide more reliable results than 2D ones. Accordingly, anti-cancer drug screenings and toxicology studies based on multicellular 3D biological models, the so-called "-oids" (e.g. spheroids, tumoroids, organoids), are blooming in the literature. However, the complex nature of these systems limit the manual quantitative analyses of single cells' behaviour in the culture. Accordingly, the demand for advanced software tools that are able to perform phenotypic analysis is fundamental. In this work, we describe the freely accessible tools that are currently available for biologists and researchers interested in analysing the effects of drugs/treatments on 3D multicellular-oids at a single-cell resolution level. In addition, using publicly available nuclear stained datasets we quantitatively compare the segmentation performance of 9 specific tools. (C) 2020 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.
  • Kilpeläinen, Tommi P.; Hellinen, Laura; Vrijdag, Johannes; Yan, Xu; Svarcbahs, Reinis; Vellonen, Kati-Sisko; Lambeir, Anne-Marie; Huttunen, Henri; Urtti, Arto; Wallen, Erik A. A.; Myohanen, Timo T. (2020)
    Previous studies have shown that prolyl oligopeptidase (PREP) negatively regulates autophagy and increases the aggregation of alpha-synuclein (alpha Syn), linking it to the pathophysiology of Parkinson's disease. Our earlier results have revealed that the potent small molecular PREP inhibitor KYP-2047 is able to increase autophagy and decrease dimerization of alpha Syn but other PREP inhibitors have not been systematically studied for these two protein-protein interaction mediated biological functions of PREP. In this study, we characterized these effects for 12 known PREP inhibitors with IC50-values ranging from 0.2 nM to 1010 nM. We used protein-fragment complementation assay (PCA) to assess alpha Syn dimerization and Western Blot of microtubule-associated protein light chain 3B II (LC3B-II) and a GFP-LC3-RFP expressing cell line to study autophagy. In addition, we tested selected compounds in a cell-free alpha Syn aggregation assay, native gel electrophoresis, and determined the compound concentration inside the cell by LC-MS. We found that inhibition of the proteolytic activity of PREP did not predict decreased alpha Syn dimerization or increased autophagy, and we also confirmed that this result did not simply reflect concentration differences of the compounds inside the cell. Thus, PREP ligands regulate the effect of PREP on autophagy and alpha Syn aggregation through a conformational stabilization of the enzyme that is not equivalent to inhibiting its proteolytic activity.
  • Lebeer, Sarah; Claes, Ingmar J. J.; Balog, Crina I. A.; Schoofs, Geert; Verhoeven, Tine L. A.; Nys, Kris; von Ossowski, Ingemar; de Vos, Willem M.; Tytgat, Hanne L. P.; Agostinis, Patrizia; Palva, Airi; Van Damme, Els J. M.; Deelder, Andre M.; De Keersmaecker, Sigrid C. J.; Wuhrer, Manfred; Vanderleyden, Jos (2012)
  • Ballhausen, Alexej; Przybilla, Moritz Jakob; Jendrusch, Michael; Haupt, Saskia; Pfaffendorf, Elisabeth; Seidler, Florian; Witt, Johannes; Hernandez Sanchez, Alejandro; Urban, Katharina; Draxlbauer, Markus; Krausert, Sonja; Ahadova, Aysel; Kalteis, Martin Simon; Pfuderer, Pauline L.; Heid, Daniel; Stichel, Damian; Gebert, Johannes; Bonsack, Maria; Schott, Sarah; Blaeker, Hendrik; Seppälä, Toni; Mecklin, Jukka-Pekka; Ten Broeke, Sanne; Nielsen, Maartje; Heuveline, Vincent; Krzykalla, Julia; Benner, Axel; Riemer, Angelika Beate; von Knebel Doeberitz, Magnus; Kloor, Matthias (2020)
    The immune system can recognize and attack cancer cells, especially those with a high load of mutation-induced neoantigens. Such neoantigens are abundant in DNA mismatch repair (MMR)-deficient, microsatellite-unstable (MSI) cancers. MMR deficiency leads to insertion/deletion (indel) mutations at coding microsatellites (cMS) and to neoantigen-inducing translational frameshifts. Here, we develop a tool to quantify frameshift mutations in MSI colorectal and endometrial cancer. Our results show that frameshift mutation frequency is negatively correlated to the predicted immunogenicity of the resulting peptides, suggesting counterselection of cell clones with highly immunogenic frameshift peptides. This correlation is absent in tumors with Beta-2-microglobulin mutations, and HLA-A*02:01 status is related to cMS mutation patterns. Importantly, certain outlier mutations are common in MSI cancers despite being related to frameshift peptides with functionally confirmed immunogenicity, suggesting a possible driver role during MSI tumor evolution. Neoantigens resulting from shared mutations represent promising vaccine candidates for prevention of MSI cancers. DNA mismatch repair (MMR)-deficient cancers with microsatellite-instability are characterized by a high load of frameshift mutation-derived neoantigens. Here, by mapping the frameshift mutation landscape and predicting the immunogenicity of the resulting peptides, the authors show evidence of immunoediting in MMR-deficient colorectal and endometrial cancers.
  • Kelloniemi, Annina; Aro, Jani; Näpänkangas, Juha; Koivisto, Elina; Mustonen, Erja; Ruskoaho, Heikki; Rysä, Jaana (2015)
    Background: The transforming growth factor (TGF)-beta is one of the key mediators in cardiac remodelling occurring after myocardial infarction (MI) and in hypertensive heart disease. The TGF-beta-stimulated clone 22 (TSC-22) is a leucine zipper protein expressed in many tissues and possessing various transcription-modulating activities. However, its function in the heart remains unknown. Methods: The aim of the present study was to characterize cardiac TSC-22 expression in vivo in cardiac remodelling and in myocytes in vitro. In addition, we used TSC-22 gene transfer in order to examine the effects of TSC-22 on cardiac gene expression and function. Results: We found that TSC-22 is rapidly up-regulated by multiple hypertrophic stimuli, and in post-MI remodelling both TSC-22 mRNA and protein levels were up-regulated (4.1-fold, P <0.001 and 3.0-fold, P <0.05, respectively) already on day 1. We observed that both losartan and metoprolol treatments reduced left ventricular TSC-22 gene expression. Finally, TSC-22 overexpression by local intramyocardial adenovirus-mediated gene delivery showed that TSC-22 appears to have a role in regulating collagen type III alpha 1 gene expression in the heart. Conclusions: These results demonstrate that TSC-22 expression is induced in response to cardiac overload. Moreover, our data suggests that, by regulating collagen expression in the heart in vivo, TSC-22 could be a potential target for fibrosis-preventing therapies.
  • Mäkitie, Riikka E.; Hackl, Matthias; Weigl, Moritz; Frischer, Amelie; Kämpe, Anders; Costantini, Alice; Grillari, Johannes; Mäkitie, Outi (2020)
    Plastin 3 (PLS3), encoded byPLS3, is a newly recognized regulator of bone metabolism, and mutations in the encoding gene result in severe childhood-onset osteoporosis. Because it is an X chromosomal gene,PLS3mutation-positive males are typically more severely affected whereas females portray normal to increased skeletal fragility. Despite the severe skeletal pathology, conventional metabolic bone markers tend to be normal and are thus insufficient for diagnosing or monitoring patients. Our study aimed to explore serum microRNA (miRNA) concentrations in subjects with defective PLS3 function to identify novel markers that could differentiate subjects according to mutation status and give insight into the molecular mechanisms by which PLS3 regulates skeletal health. We analyzed fasting serum samples for a custom-designed panel comprising 192 miRNAs in 15 mutation-positive (five males, age range 8-76 years, median 41 years) and 14 mutation-negative (six males, age range 8-69 years, median 40 years) subjects from four Finnish families with differentPLS3mutations. We identified a unique miRNA expression profile in the mutation-positive subjects with seven significantly upregulated or downregulated miRNAs (miR-93-3p, miR-532-3p, miR-133a-3p, miR-301b-3p, miR-181c-5p, miR-203a-3p, and miR-590-3p;pvalues, range .004-.044). Surprisingly, gender subgroup analysis revealed the difference to be even more distinct in female mutation-positive subjects (congruentpvalues, range .007-.086) than in males (pvalues, range .127-.843) in comparison to corresponding mutation-negative subjects. Although the seven identified miRNAs have all been linked to bone metabolism and two of them (miR-181c-5p and miR-203a-3p) have bioinformatically predicted targets in thePLS33 ' untranslated region (3 '-UTR), none have previously been reported to associate with PLS3. Our results indicate thatPLS3mutations are reflected in altered serum miRNA levels and suggest there is crosstalk between PLS3 and these miRNAs in bone metabolism. These provide new understanding of the pathomechanisms by which mutations inPLS3lead to skeletal disease and may provide novel avenues for exploring miRNAs as biomarkers in PLS3 osteoporosis or as target molecules in future therapeutic applications. (c) 2020 The Authors.Journal of Bone and Mineral Researchpublished by American Society for Bone and Mineral Research.