RESUMO
Bone matrix formation and mineralization are two closely related, yet separated processes. Matrix formation occurs first, mineralization is a second step strictly dependent on the dietary intake of calcium and phosphorus (P). However, mineralization is commonly used as diagnostic parameter for bone-related diseases. In this context, bone loss, often characterized as a condition with reduced bone mineral density, represents a major burden for human health, for which increased dietary mineral intake is generally recommended. Using a counterintuitive approach, we use a low-P diet followed by a sufficient-P intake to increase bone volume. We show in zebrafish by histology, qPCR, micro-CT, and enzyme histochemistry that a two-months period of reduced dietary P intake stimulates extensive formation of new bone matrix, associated with the upregulation of key genes required for both bone matrix formation and mineralization. The return to a P-sufficient diet initiates the mineralization of the abundant matrix previously deposited, thus resulting in a striking increase of the mineralized bone volume as proven at the level of the vertebral column, including vertebral bodies and arches. In summary, bone matrix formation is first stimulated with a low-P diet, and its mineralization is later triggered by a sufficient-P dietary intake. In zebrafish, the uncoupling of bone formation and mineralization by alternating low and sufficient dietary P intake significantly increases the bone volume without causing skeletal malformations or ectopic mineralization. A modification of this approach to stimulate bone formation, optimized for mammalian models, can possibly open opportunities to support treatments in patients that suffer from low bone mass.
RESUMO
Introduction: Trimeric intracellular potassium channels TRIC-A and -B are endoplasmic reticulum (ER) integral membrane proteins, involved in the regulation of calcium release mediated by ryanodine (RyRs) and inositol 1,4,5-trisphosphate (IP3Rs) receptors, respectively. While TRIC-A is mainly expressed in excitable cells, TRIC-B is ubiquitously distributed at moderate level. TRIC-B deficiency causes a dysregulation of calcium flux from the ER, which impacts on multiple collagen specific chaperones and modifying enzymatic activity, leading to a rare form of osteogenesis imperfecta (OI Type XIV). The relevance of TRIC-B on cell homeostasis and the molecular mechanism behind the disease are still unknown. Results: In this study, we exploited zebrafish to elucidate the role of TRIC-B in skeletal tissue. We demonstrated, for the first time, that tmem38a and tmem38b genes encoding Tric-a and -b, respectively are expressed at early developmental stages in zebrafish, but only the latter has a maternal expression. Two zebrafish mutants for tmem38b were generated by CRISPR/Cas9, one carrying an out of frame mutation introducing a premature stop codon (tmem38b-/- ) and one with an in frame deletion that removes the highly conserved KEV domain (tmem38bΔ120-7/Δ120-7 ). In both models collagen type I is under-modified and partially intracellularly retained in the endoplasmic reticulum, as described in individuals affected by OI type XIV. Tmem38b-/- showed a mild skeletal phenotype at the late larval and juvenile stages of development whereas tmem38bΔ120-7/Δ120-7 bone outcome was limited to a reduced vertebral length at 21 dpf. A caudal fin regeneration study pointed towards impaired activity of osteoblasts and osteoclasts associated with mineralization impairment. Discussion: Our data support the requirement of Tric-b during early development and for bone cell differentiation.
Assuntos
Canais Iônicos , Osteogênese Imperfeita , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Osso e Ossos/metabolismo , Cálcio/metabolismo , Diferenciação Celular/genética , Canais Iônicos/genética , Osteogênese Imperfeita/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Osteogenesis imperfecta (OI) is a group of heritable disorders affecting bone and other connective tissues. Dominant OI forms are mainly caused by mutations in collagen type I. Patients suffer from skeletal deformities, fractures of long bones and vertebral compression fractures from early childhood onward. Altered collagen structure and excess mineralisation are the main causes for the bone phenotype. The Chihuahua (Chi/+) zebrafish has become an important model for OI. Given that reduced dietary phosphorus (P) intake reduces the bone mineral content and promotes bone matrix formation in teleosts, including zebrafish, we tested whether a low dietary P (LP) intake mitigates the OI phenotype in the Chi/+ model. To answer this question, we characterised the Chi/+ vertebral column phenotype at a morphological, cellular and subcellular level. We present the first description of vertebral compression fractures in Chi/+ and assess the effects of LP diet on the Chi/+ phenotype (Chi/+LP). Compared to untreated Chi/+, two months of LP dietary treatment decreases vertebral deformities in the abdominal region and reduces shape variation of caudal vertebral bodies to a condition more similar to wild type (WT). At the histological level, the osteoid layer, covering the bone at the vertebral body endplates in WT zebrafish, is absent in Chi/+, but it is partially restored with the LP diet. Whole mount-stained specimens and histological sections show various stages of vertebral compression fractures in Chi/+ and Chi/+LP animals. Both Chi/+ and Chi/+LP show abundant osteoclast activity compared to WT. Finally, the ultrastructure analysis of WT, Chi/+ and Chi/+LP shows Chi/+ and Chi/+LP osteoblasts with enlarged endoplasmic reticulum cisternae and a high protein content, consistent with intracellular retention of mutated collagen. Nevertheless, the secreted collagen in Chi/+LP appears better organised concerning fibre periodicity compared to Chi/+. Our findings suggest that a reduced mineral content of Chi/+ bone could explain the lower frequency of vertebral column deformities and the restored shape of the vertebral bodies in Chi/+LP animals. This, together with the improved quality of the bone extracellular matrix, suggests that two months of reduced dietary P intake can alleviate the severe bone phenotype in Chi/+ zebrafish.
Assuntos
Fraturas por Compressão , Anormalidades Musculoesqueléticas , Osteogênese Imperfeita , Fraturas da Coluna Vertebral , Animais , Colágeno , Dieta , Modelos Animais de Doenças , Humanos , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/patologia , Fenótipo , Fósforo , Peixe-ZebraRESUMO
Dietary phosphorus (P) is essential for bone mineralisation in vertebrates. P deficiency can cause growth retardation, osteomalacia and bone deformities, both in teleosts and in mammals. Conversely, excess P supply can trigger soft tissue calcification and bone hypermineralisation. This study uses a wide range of complementary techniques (X-rays, histology, TEM, synchrotron X-ray tomographic microscopy, nanoindentation) to describe in detail the effects of dietary P on the zebrafish skeleton, after two months of administering three different diets: 0.5% (low P, LP), 1.0% (regular P, RP), and 1.5% (high P, HP) total P content. LP zebrafish display growth retardation and hypomineralised bones, albeit without deformities. LP zebrafish increase production of non-mineralised bone matrix, and osteoblasts have enlarged endoplasmic reticulum cisternae, indicative for increased collagen synthesis. The HP diet promotes growth, high mineralisation, and stiffness but causes vertebral centra fusions. Structure and arrangement of bone matrix collagen fibres are not influenced by dietary P in all three groups. In conclusion, low dietary P content stimulates the formation of non-mineralised bone without inducing malformations. This indicates that bone formation and mineralisation are uncoupled. In contrast, high dietary P content promotes mineralisation and vertebral body fusions. This new zebrafish model is a useful tool to understand the mechanisms underlying osteomalacia and abnormal mineralisation, due to underlying variations in dietary P levels.