Syndecan-3 enhances anabolic bone formation through WNT signalling

Brito, Francesca Manuela Johnson de Sousa; Butcher, Andrew; Pisconti, Addolorata; Poulet, Blandine; Prior, Amanda; Charlesworth, Gemma; Sperinck, Catherine; Mase, Michele Scotto di; Bou‐Gharios, George; Hof, Robert Jurgen van 't; Daroszewska, Anna

doi:10.1101/846972

Cited by 1 publication

(7 citation statements)

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“…NRecon (Bruker) and DataViewer (Bruker) were used to construct cross-sectional slices or to generate 3D computational models, respectively. CTAn (Bruker) was used to quantify metrics, essentially as previously described (71). For trabecular tibial and femoral measurements, a volume of interest (VOI) encompassing the region 90-990 µm below or above (respectively) the growth plates were used.…”

Section: Micro-computed Tomography (μCt)mentioning

confidence: 99%

“…After analysis by μCT (above), the tibiae were in methyl methacrylate (MMA) and 5 µm sections cut with a tungsten steel knife on a motorised rotary microtome (Leica). Sections were stained either with Goldner's Trichrome to analyse osteoid and lipid droplets or counterstained with calcein blue to quantify the amount of bone formed between the injected alazarin red-S and calcein, essentially as previously described (71). Images were captured using an Axioscan Z1 slide scanner (Zeiss) and histomorphometry performed using the OsteoidHisto and CalceinHisto open-source image analysis programs (25).…”

Section: Histology and Histomorphometrymentioning

confidence: 99%

“…Osteoblasts were isolated from the calvariae of 3-day-old mouse pups essentially as previously described (71). Briefly, dissected calvariae were digested with collagenase type I (Sigma-Aldrich) and the isolated cells cultured in α-MEM (Gibco) supplemented with 2 mM L-glutamine, 10% FBS, and antibiotics.…”

Section: Osteoblast and Haematopoietic Stromal Cell Isolation And Cul...mentioning

confidence: 99%

“…A separate population of osteoblasts and haematopoietic stromal cells were isolated from leg bone chips and marrow respectively, dissected from the long bones of 20-week-old mice. The epiphyses were removed to expose the marrow, which was flushed from the bones by centrifugation and cultured directly with α-MEM (supplemented as above but also with 100 ng/ml M-CSF for 3 days to obtain M-CSF-dependent bone marrow macrophages (BMMs) (71)). The remaining bone was cut into small chips of approximately 2 mm 3 and collagenase digested for 1 hour.…”

Section: Osteoblast and Haematopoietic Stromal Cell Isolation And Cul...mentioning

confidence: 99%

“…For alkaline phosphatase assays the cells were formalin-fixed, freeze-thawed and incubated with 20mM paranitrophenyl phosphate (PNPP (ThermoFisher Scientific) which is dephosphorylated by alkaline phosphatase to produced paranitrophenol (PNP), the absorbance of which was measured at 405 nm after 30 minutes. PNP levels were normalised against cell proliferation measurements, all essentially as described (71).…”

Section: Osteoblast Proliferation and Alkaline Phosphatase Assaysmentioning

confidence: 99%

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miR-324 mediates bone homeostasis through the regulation of osteoblast and osteoclast differentiation and activity

Hayman

Lin

Prior

et al. 2023

Preprint

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microRNAs (miRNAs) are non-coding RNAs which modulate the expression of other RNA molecules. One miRNA can target many transcripts, allowing each miRNA to play key roles in many biological pathways. miR-324 is a miRNA previously implicated in bone and cartilage maintenance, defects of which result in common age-related diseases, such as osteoporosis or osteoarthritis (OA). In global miR-324-null mice cartilage damage was increased in both surgically and ageing-induced OA, despite minimal changes to the cartilage transcriptome, with few predicted miR-324 targets dysregulated. However, micro-computed tomography and histology demonstrated that global miR-324-null the mice had an increase in bone mineral density, trabecular thickness and cortical thickness, with many parameters increasing with age. The bone marrow of miR-324-null mice also had reduced lipid content while and in vivo TRAP staining revealed a decrease in osteoclasts, with histomorphometry demonstrating an increased rate of bone formation in miR-324-null mice. Ex vivo assays revealed that the high bone mass phenotype of the miR-324-null mice resulted from increased osteoblast activity and decreased osteoclastogenesis. RNA-seq and qRT-PCR followed by miR-324 target prediction and validation in osteoblasts, osteoclasts and bone marrow macrophages identified the osteoclast fusion regulator Pin1 as a miR-324 target in the osteoclast lineage and the master osteogenic regulator Runx2 as a target of miR-324-5p in osteoblasts, the in vitro overexpression of which recapitulated the increased osteogenesis and decreased adipogenesis phenotype observed in vivo. These data point to important roles of miR-324 in skeletal biology with altered bone homeostasis in miR-324-null mice potentially causal for the increased cartilage damage observed during OA and ageing. Elucidation of pathways regulated by miR-324 offer promise for the treatment of bone diseases such as osteoporosis.

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Section: Micro-computed Tomography (μCt)mentioning

confidence: 99%

Section: Histology and Histomorphometrymentioning

confidence: 99%

Section: Osteoblast and Haematopoietic Stromal Cell Isolation And Cul...mentioning

confidence: 99%

Section: Osteoblast and Haematopoietic Stromal Cell Isolation And Cul...mentioning

confidence: 99%

Section: Osteoblast Proliferation and Alkaline Phosphatase Assaysmentioning

confidence: 99%

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