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Exploring the impact of energy metabolism in periosteal cells transcriptional profile.

ABSTRACT: Glycolytic metabolism, once considered merely a consequence of cellular function, has emerged as a crucial regulator of cellular differentiation. Both glycolysis and oxidative phosphorylation (OXPhos) are vital for osteoblastogenesis. Osteoblasts are the cells that make bone. To deepen our understanding of OXPhos's role in bone mass accrual, we engineered a mutant mouse model deficient in Mitochondrial Transcription Factor A (TFAM) specifically in mesenchymal progenitors within the limb bud and their descendants, using the PRX1-Cre driver system. TFAM is essential for transcription of the mitochondrial genes that encode critical components of the electron transport chain, thereby governing OXPhos. Our TFAM mutant mice exhibit marked shortening of long bones, evident at birth and progressing with age, accompanied by growth plate abnormalities and bone deformities. Critically, these mice develop spontaneous fractures in long bone mid-shafts by three weeks of age, indicating significant compromise in bone integrity. Comparative analyses using MicroCT and histomorphometry reveal drastic cortical bone thinning in mutants due to severely impaired osteoid deposition and bone formation, especially in the diaphyseal periosteum. Furthermore, an increased number of osteoclasts at this site suggests that an elevated bone resorption contributes to the phenotype. Acknowledging the active role of energy metabolism in cell differentiation and function, we measured ATP production in PRX1 lineage periosteal cells. TFAM deletion led to a substantial decrease in steady-state intracellular ATP levels, highlighting a critical metabolic deficit. To rectify this metabolic defect and potentially correct the resultant phenotype, we bred TFAM mice with another mouse line expressing mutated, oxygen-independent Hypoxia Inducible Factor 1alpha (HIF1dPA) within PRX1 lineage cells (yielding TFAM/HIF1dPA mice). These double mutants did not experience spontaneous fractures, and their cortical bone thickness and the ability of periosteal cells to accumulate osteoid was fully restored. Therefore, by examining the transcriptional profile of periosteal cells, our aim is to determine whether TFAM deficiency alters their genetic landscape and if HIF1 activation can reverse these changes. Our goal is to provide new insights into the role of energy metabolism in shaping the transcriptome landscape, thereby influencing cell differentiation and activity.

ORGANISM(S): Mus musculus

PROVIDER: GSE261114 | GEO | 2024/08/18

REPOSITORIES: GEO

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Project description:The expression of Prx1 has been used as a marker to define the skeletal stem cells (SSC) populations found within the bone marrow and periosteum that contribute to bone regeneration. However, Prx1 expressing SSCs (Prx1-SSCs) are not restricted to the bone compartments, but are also located within the muscle and able to contribute to ectopic bone formation. Little is known however, about the mechanism(s) regulating Prx1-SSCs that reside in muscle and how they participate in bone regeneration. This study compared both the intrinsic and extrinsic factors of the periosteum and muscle derived Prx1-SSCs and analyzed their regulatory mechanisms of activation, proliferation, and skeletal differentiation. There was considerable transcriptomic heterogeneity in the Prx1-SSCs found in muscle or the periosteum however in vitro cells from both tissues show tri-lineage (adipose, cartilage and bone) differentiation. At homeostasis, periosteal derived Prx1 cells were proliferative and low levels of BMP2 were able to promote their differentiation, while the muscle derived Prx1 cells were quiescent and refractory to comparable levels of BMP2 that promoted periosteal cell differentiation. The transplantation of Prx1-SCC from muscle and periosteum into either the same site from which they were isolated, or their reciprocal sites showed that periosteal cell transplanted onto the surface of bone tissues differentiated into bone and cartilage cells but was incapable of similar differentiation when transplanted into muscle. Prx1-SSCs from the muscle showed no ability to differentiate at either site of transplantation. Both fracture and ten times the BMP2 dose was needed to promote muscle-derived cells to rapidly enter the cell cycle as well as undergo skeletal cell differentiation. This study elucidates the diversity of the Prx1-SSC population showing that cells within different tissue sites are intrinsically different. While muscle tissue must have factors that promote Prx1-SSC to remain quiescent, either bone injury or high levels of BMP2 can activate these cells to both proliferate and undergo skeletal cell differentiation. Finally, these studies raise the possibility that muscle SSCs are potential target for skeletal repair and bone diseases.

Project description:Vitamin A is the only known compound that produces spontaneous fractures in rats. In an effort to resolve the molecular mechanism behind this effect, we fed young rats high doses of vitamin A and performed a global transcriptional analysis of diaphyseal bone after one week, i.e. just before the first fractures appeared. Microarray gene expression analysis revealed that 68 transcripts were differentially expressed in hypervitaminotic cortical bone and 118 transcripts were found when the bone marrow was also included. 98% of the differentially expressed genes in the bone marrow sample were up-regulated. In contrast, hypervitaminotic cortical bone without marrow showed reduced expression of 37% of differentially expressed genes. Gene Ontology (GO) analysis revealed that only samples containing bone marrow were associated to a GO term, which principally represented extracellular matrix (ECM). This is consistent with the histological findings of increased endosteal bone formation. Four of the genes in this ECM cluster and four other genes, including Cyp26b1 which is known to be up-regulated by vitamin A, were selected and verified by real-time PCR. In addition, immunohistochemical staining of bone sections confirmed that the bone-specific molecule, osteoadherin (Omd) was up-regulated. Further analysis of the major gene expression changes revealed distinct differences between cortical bone and bone marrow, e.g. there appeared to be augmented Wnt signaling in the bone marrow but reduced Wnt signaling in cortical bone. Moreover, induced expression of hypoxia-associated genes was only found in samples containing bone marrow. Together, these results corroborate our previous observations of compartment-specific effects of vitamin A, with reduced periosteal but increased endosteal bone formation, and suggest important roles for Wnt signaling and hypoxia in the processes leading to spontaneous fractures. Rat diaphyseal bones from hypervitaminosis A and controls (8week old) were extracted for RNA and hybridized on Affymetrix microarrays. Humeri were isolated, and all connective tissue, including periosteum, was completely removed as were both epiphyses, including growth plates. Samples of cortical bone including marrow were snap-frozen in liquid nitrogen and quickly crushed into a fine powder using a mortar and pestle followed by total RNA extraction with TRI Reagent® (Sigma-Aldrich). For cortical bone without marrow, diaphyseal bones were cut into pieces, vortexed in ice-cold PBS three times for 10 s to remove marrow cells followed by snap-freezing in liquid nitrogen, crushing into a fine powder and RNA extraction. Isolated RNA was quantified using spectrophotometry by measuring the absorbance at 260 nm, and the 260/280 nm ratio was calculated. RNA was kept only when this ratio was 1.9-2.0 to ensure the absence of protein contamination and limited RNA degradation. The integrity of sample RNAs was confirmed by capillary electrophoresis separating 18 S and 28 S ribosomal RNA on an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Notably, the metaphyses and growth plates were not included in theses samples. Importantly the amount of RNA extracted from cortical bone including marrow was approximately four times the amount of RNA extracted from the pure cortical bone and was therefore considered herein to mainly represent bone marrow cells.

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Exploring the impact of energy metabolism in periosteal cells transcriptional profile.

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