Loss of coactosin-like F-actin binding protein 1 (Cotl1) decreases platelet-mediated osteoclastogenesis and causes osteopetrosis phenotypes in mouse

BACKGROUNDSOsteopetrosis, a rare skeletal disease, is characterized by an increased bone mass resulting from impaired bone remodeling process. Platelet is the major bone-healing blood component involved in the regulation of bone resorption, particularly in the removal of compromised bones. Several actin-associated proteins contribute to the orchestration of actin ring formation in osteoclasts closely related to bone resorption. However, the role of coactosin-like F-actin binding protein 1 (Cotl1) in actin ring formation and platelet-mediated bone resorption mechanisms remains unclear. METHODSWhole-mount in situ RNA hybridization was performed to detect cotl1 expression pattern in zebrafish. cotl1 gene knockdown zebrafish using morpholino oligonucleotides and platelet marker-expressing transgenic zebrafish were investigated for finding the phenotypic clues. Cotl1 knockout (Cotl1-/-) mice were generated using Cre/loxP recombination systems. In silico network analysis of the differentially expressed genes between bone marrow samples of wild type and Cotl1-/- mice was conducted. Primary-cultured monocytes from Cotl1-/- mice were examined for osteoclast differentiation and mRNA and protein expression patterns. Cotl1-/- mice underwent hematological examination and bone phenotype assessments including micro-CT, bone density, histology, immunohistochemistry, electron microscopy, and mechanical testing. Genetic association of SNPs in human COTL1 gene with estimated bone mineral density was analyzed. RESULTSZebrafish cotl1 mRNA was highly expressed in the caudal hematopoietic tissue region. Knockdown of cotl1 in zebrafish embryos decreased the expression of c-myb, a marker of hematopoietic stem cells (HSCs). Notably, the platelet receptor CD41 was reduced in the HSCs of cotl1-depleted zebrafish and Cotl1-/- mice showed reduced platelet production with platelet surface markers of CD41 and CD61. Significantly reduced osteoclast differentiation and bone resorption pit, and impaired actin ring formation were observed in the primary myocytes from Cotl1-/- mice. Structural and histological analyses of the femur revealed sclerotic bone phenotypes in Cotl1-/- mice. Mechanical assessment of Cotl1-/- mouse femoral bones revealed osteopetrotic phenotypes. Association analysis of genetic variants in COTL1 gene in subjects from the UK Biobank suggested that COTL1 is susceptible to bone density in humans. CONCLUSIONSOur results provide insights into the role of Cotl1 in platelet-mediated osteoclastogenesis and the novel finding that the loss of Cotl1-/- mice causes osteopetrosis phenotypes. Clinical PerspectiveWhat Is New? O_LIDeficiency of Cotl1 decreased platelet production in zebrafish and mice. C_LIO_LIAbsence of Cotl1 disrupted the actin ring formation which is crucial for osteoclast differentiation in bone remodeling process. C_LIO_LICotl1 knockout mice displayed sclerotic bone phenotypes and increased bone density that are representative characteristics of osteopetrosis. C_LIO_LIGenetic variants in COTL1 gene in subjects from the UK Biobank are significantly associated with bone density. C_LI What Are the Clinical Implications? O_LIThe current findings suggest that Cotl1 plays a fundamental role in platelet production-mediated osteoclastogenesis during bone remodeling, providing valuable insights into novel strategies for bone health maintenance. C_LIO_LICotl1 may be a promising target for novel therapeutic strategies for the treatment and/or prevention of impaired osteoclastogenesis-mediated bone diseases such as osteopetrosis and osteoporosis. C_LI