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Serca2 deletion in the mouse adult Bmi1+ compartment induces a lethal phenotype involving a severe gastric dysfunction
Diego Herrero, Antonio Docavo, María Salvador, Marina Higuera, Alfonso Cortés, Javier García-Ceca, Sara Montero-Herradón, Miguel A García-Brenes, Guillermo Albericio, Alejandra Cordero, Rosa M Carmona, Antonio de Molina, Jesús M Salvador, Carmen Mora, Agustín G Zapata, Antonio Bernad
Diego Herrero, Antonio Docavo, María Salvador, Marina Higuera, Miguel A García-Brenes, Guillermo Albericio, Alejandra Cordero, Jesús M Salvador, Antonio Bernad, Department of Immunology and Oncology, National Center for Biotechnology (CNB.CSIC), Madrid 28049, Spain
Alfonso Cortés, Center of Cytometry and Fluorescence Microscopy, Complutense University, Madrid 28040, Spain
Javier García-Ceca, Sara Montero-Herradón, Agustín G Zapata, Health Research Institute, Hospital 12 de Octubre (imas12), Madrid 28041, Spain
Javier García-Ceca, Sara Montero-Herradón, Agustín G Zapata, Department of Cell Biology and Histology, Faculty of Biological Sciences, Complutense University of Madrid, Madrid 28040, Spain
Rosa M Carmona, Department of Cardiovascular, National Center for Cardiovascular Research, Madrid 28049, Spain
Antonio de Molina, Department of CNIC Histopathology, National Center for Cardiovascular Research, Madrid 28029, Spain
Carmen Mora, Department of Business, MedSir Org, Madrid 28049, Spain
Co-first authors: Diego Herrero and Antonio Docavo.
Co-corresponding authors: Agustín G Zapata and Antonio Bernad.
Author contributions: Herrero D and Docavo A performed the main block of experiments, collected data and performed data analysis; Salvador M, Higuera M, García-Brenes MA, Cortés A, García-Ceca J, Montero-Herradón S and Roche E collaborated in specific aspects of the research program, collected data and performed data analysis; Cordero A and Carmona RM has been the main responsible for the coordination of the different mouse colonies and genotyping; Albericio G and Higuera M have supervised different experimental aspects of the project; Bernad A conceived the project, designed the global strategy and supervised research; Salvador JM and Mora C have co-coordinated the lymphohematopoietic analysis of the KO mice; Zapata AG, García-Ceca J, Montero-Herradón S have been the main responsible of the dissection of the thymic phenotype; Bernad A wrote and edited the manuscript with the main collaboration of Higuera M and Zapata AG; Herrero D, Docavo A, Salvador M, Higuera M, Cortés A, García-Ceca J, Montero-Herradón S, García-Brenes MA, Albericio G, Cordero A, Carmona RM, de Molina A, Salvador JM, Mora C, Zapata AG, Bernad A read and approved the final manuscript.
Supported by the Spanish Ministry of Science and Innovation, AEI/FEDER, UE, No. RTI2018-097604-B-I00; The Instituto de Salud Carlos III, Cell Therapy Network, TERCEL, No. RD16/0011/0037; The Spanish Ministry of Science, Innovation and Universities, No. RTI2018-093938-B-I00; The Carlos III Health Institute, Cell Therapy Network, TERCEL, No. RD16/0011/0002; and The program has been funded by the Regional Government of Madrid Program, Avancell, No. B2017/BMD-3692.
Institutional review board statement: This study did not involve human participants, consequently, institutional review board statement and approval, and informed consent procedures were not applicable. The project (RTI2018-097604-B-I00) has been evaluated (Ref. REGAGE25e00055149413) by the specific panels for Biomedicine of the Spanish Ministry of Science and Innovation and the Cell Therapy of the National Healthy Institute, Carlos III (RD16/0011/0037) and the Ethical CSIC Committee. These evaluations involved the argumentation on the use of specific human cells and a detailed justification of the animal work.
Institutional animal care and use committee statement: Animal studies were approved (Ref. 23002) by the Ethics Committee of the National Center for Biotechnology (CEEA-CNB).
Conflict-of-interest statement: The authors declare that they have no conflict of interest.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Data sharing statement: Authors be willing to distribute any materials and protocols used in the published experiments to qualified researchers for their own use. These must be made available with minimal restrictions and in a timely manner. Specific data sets must be made freely available to readers from the date of publication upon request.
Corresponding author: Antonio Bernad, MD, Doctor, Department of Immunology and Oncology, National Center for Biotechnology (CNB.CSIC), C/Darwin 3 Universidad Autónoma de Madrid Campus de Cantoblanco, Madrid 28049, Spain.
abernad@cnb.csic.es
Received: August 13, 2025
Revised: October 28, 2025
Accepted: February 25, 2026
Published online: May 21, 2026
Processing time: 278 Days and 23 Hours
BACKGROUND
Cytoplasmic calcium ions (Ca2+) are well-known intracellular signalling molecules, being involved in the integration of a variety of metabolic, proliferative and survival signals. They have been demonstrated an important role in embryonic and most adult stem cells (ASC). In the gut, it has been long recognized that Ca2+ handling is critical for the generation of pacemaker activity by Cajal interstitial cells. More recently, some studies in have shown that cytosolic Ca2+ plays a central role in intestinal stem cell (ISC) regulation, while this connection remains undefined in mammalian ISC populations.
AIM
To evaluate whether cytosolic Ca2+ could play an equivalent role in murine ISC population as in Drosophila melanogaster, and compare this effect to the lymphohematopoietic compartment.
METHODS
To this aim, we disrupted the expression of the calcium transporter Serca2, one of the crucial players in Ca2+ storage, within murine ISC population. Murine ISC compartment was selected using Bmi1, a well-established marker for reserve ISCs crucial in regeneration upon injury, and Serca2 conditional deletion was evaluated in the gut epithelium using the Serca2Bmi1homo mouse strain. Main results were compared with the Serca2villinhomo strain. Metabolic parameters were evaluated, including food and water intake, body weight evolution, glucose response, serum biochemical markers and fecal fat content. Histological analysis along the gastric tract was used to assess Bmi1+ ISC proliferative state. Intestinal crypts were purified and analyzed for proliferation, cell death and gene expression. Total and segmented intestine was analyzed for differential gene expression. Due to the severe phenotype found, the impact of Serca2 conditional deletion was later evaluated in the lymphohematopoietic system, to eliminate a generalized effect.
RESULTS
Conditional deletion of Serca2 in Bmi1+ adult ASC (DSerca2Bmi1homo mice) resulted, unexpectedly, in a lethal phenotype. Important weight loss suggested severe malabsorption/malnutrition as the most probable cause of deaths. Results were further confirmed in DSerca2Villin mice, that developed a fulminant phenotype during the first week after tamoxifen-induction. Blood biochemistry showed a reduced glucose uptake, while feces content showed an increased total amount of fat in DSerca2Villin mice. Histological analysis along gastric tract revealed major extension of Bmi1+ cell compartment in DSerca2Bmi1homo mice. In the gut, Bmi1+ ISC population showed a substantial increase (3-fold) in their proliferative rate upon Serca2 deletion. Differential gene expression did not identify major changes in intestinal crypts, but key calcium regulatory players such as Orai-1, Pmca-1 and Pmca-4 expression were affected, as well as endoplasmic reticulum stress response genes. Finally, important thymic alterations were characterized in DSerca2Bmi1 mice, plausibly associated to the malnourished state of the animals.
CONCLUSION
As in Drosophila melanogaster, cytosolic Ca2+ is a key element of murine ISC regulation, affecting their proliferation rate and the proper differentiation equilibrium. Severe lethality provoked by Serca2 deletion in both gut Bmi1+ and Villin+ compartments raises the possibility that dysregulation of cytosolic Ca2+ could play some role in some gastric pathologies.
Core Tip: Although cytoplasmic [calcium ions (Ca2+)] plays an important role in regulating several critical cellular processes, low information is available regarding intestinal stem cells (ISC). Recent seminal results, obtained in Drosophila melanogaster, have demonstrated that the reduction of Ca2+ in ISC increases their proliferation rate; downregulation of Serca is one of the main genes involved. Based on these relevant results, and taking advantage of a dedicated mouse models, we have demonstrated that the deletion of Serca2 in the intestinal Bmi1+ populations provokes, unexpectedly, a lethal phenotype, associated with a severe malnutrition. Furthermore, because this promotes ISC proliferation rate a moderate reduction of Serca2 activity could be involved in some neoplasic conditions.
