Dataset Information

RNAseq and RNA molecular barcoding reveal differential gene expression in cortical bone following hindlimb unloading in female mice.

ABSTRACT: Disuse-induced bone loss is seen following spinal cord injury, prolonged bed rest, and exposure to microgravity. We performed whole transcriptomic profiling of cortical bone using RNA sequencing (RNAseq) and RNA molecular barcoding (NanoString) on a hindlimb unloading (HLU) mouse model to identify genes whose mRNA transcript abundances change in response to disuse. Eleven-week old female C57BL/6 mice were exposed to ambulatory loading or HLU for 7 days (n = 8/group). Total RNA from marrow-flushed femoral cortical bone was analyzed on HiSeq and NanoString platforms. The expression of several previously reported genes associated with Wnt signaling and metabolism was altered by HLU. Furthermore, the increased abundance of transcripts, such as Pfkfb3 and Mss51, after HLU imply these genes also have roles in the cortical bone's response to altered mechanical loading. Our study demonstrates that an unbiased approach to assess the whole transcriptomic profile of cortical bone can reveal previously unidentified mechanosensitive genes and may eventually lead to novel targets to prevent disuse-induced osteoporosis.

SUBMITTER: Spatz JM

PROVIDER: S-EPMC8509868 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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RNAseq and RNA molecular barcoding reveal differential gene expression in cortical bone following hindlimb unloading in female mice.

Spatz Jordan M JM Ko Frank C FC Ayturk Ugur M UM Warman Matthew L ML Bouxsein Mary L ML

PloS one 20211012 10

Disuse-induced bone loss is seen following spinal cord injury, prolonged bed rest, and exposure to microgravity. We performed whole transcriptomic profiling of cortical bone using RNA sequencing (RNAseq) and RNA molecular barcoding (NanoString) on a hindlimb unloading (HLU) mouse model to identify genes whose mRNA transcript abundances change in response to disuse. Eleven-week old female C57BL/6 mice were exposed to ambulatory loading or HLU for 7 days (n = 8/group). Total RNA from marrow-flushe ...[more]

PMID: 34637435

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Project description:BackgroundAn oligopeptide hepcidin is encoded by the human HAMP gene (Hamp in mice). Its deficiency can result in iron overload, while excess may lead to iron deficiency. Hepcidin knockout mice exhibited iron accumulation in multiple tissues, accompanied by degeneration of bone microarchitecture and reduced biomechanical properties. Astronauts who are exposed to weightlessness during prolonged spaceflight experience bone loss. After space missions, an interrelation exists between iron stores and bone mineral density (BMD). Bone loss in mice due to unloading is linked to iron excess and involves hepcidin. The potential role of hepcidin in unloading-induced bone loss remains unclear.MethodsOur study conducted relevant experiments using hepcidin knockout mice and their primary osteoblasts as the research subjects. We used the hindlimb unloading (HLU) model and the random positioning machine (RPM) system to simulate weightlessness in vivo and in vitro.ResultsHLU mice exhibited reduced hepcidin levels in the serum and liver. Hepcidin knockout further diminished BMD and bone mineral content (BMC) in the femurs of HLU mice. Similarly, the bone volume fraction (BV/TV) and connectivity density (Conn.Dn) followed this downward trend, whereas trabecular separation (Tb.Sp) showed an inverse pattern. Moreover, hepcidin knockout decreased the ultimate load and elastic modulus in the tibias of HLU mice. Hepcidin knockout decreased PINP levels in the serum, a commonly used marker for bone formation, alongside elevated iron levels in the serum, liver, and bone of HLU mice. We also found higher serum MDA and SOD levels in these mice. In vitro, experimental data indicated that hepcidin knockout suppresses the osteoblastic differentiation capacity under RPM conditions. Additionally, this condition upregulates SOST protein levels and downregulates LRP6 and β-catenin protein levels in osteoblasts.ConclusionHepcidin knockout exacerbates bone loss in HLU mice, most likely due to reduced osteoblastic activity.

Project description:Previous work has shown that the soluble murine BMPR1A-fusion protein (mBMPR1A-mFc) binds to BMP2 and BMP4 with high affinity, preventing downstream signaling. Further, treatment of intact and ovariectomized mice with mBMPR1A-mFc leads to increased bone mass, and improved bone microarchitecture and strength, via increased bone formation and reduced resorption. In this study, we tested the effects of mBMPR1A-mFc on disuse-induced bone loss caused by 21 days of hindlimb unloading (HLU) via tail suspension versus cage controls (CONs). Adult female C57BL/6J mice (12 weeks old) were assigned to one of four groups (n = 10 each): CON-VEH; CON-mBMPR1A-mFc; HLU-VEH; and HLU-mBMPR1A-mFc. Mice were injected subcutaneously with VEH or mBMPR1A-mFc (4.5 mg/kg, 2×/week). Leg BMD declined in the HLU-VEH group (-5.3% ± 1.3%), whereas it was unchanged in HLU-mBMPR1A-mFc (-0.3% ± 0.9%, p < 0.05 versus HLU-VEH). Leg BMD increased significantly more in CON-mBMPR1A-mFc than CON-VEH (10.2% ± 0.6% versus 4.4% ± 0.8%). In the femur, trabecular, and cortical bone microarchitecture was worse in the HLU-VEH compared to CON-VEH mice, whereas mBMPR1A-mFc treatment for 3 weeks led to greater Tb.BV/TV, Tb.Th, and midshaft Ct.Th in both the HLU and CON groups compared to comparable VEH-treated counterparts (p < 0.05). HLU-mBMPR1A-mFc mice also had 21% greater failure load (p < 0.05) compared to their VEH-treated counterparts. Dynamic histomorphometry indicated that treatment with mBMPR1A-mFc led to significantly greater mineralizing surface and mineral apposition rate, resulting in a 3.5-fold and fivefold higher bone formation rate in the mBMPR1A-mFc-treated CON and HLU animals versus VEH groups, respectively. mBMPR1A-mFc-treated mice had a similar osteoblast surface but significantly lower osteoclast surface than VEH-treated animals in both the CON and HLU groups. Altogether, these findings suggest that treatment with the soluble BMPR1A fusion protein may be useful for maintenance of skeletal integrity in the setting of disuse-induced bone loss.

Dataset Information

RNAseq and RNA molecular barcoding reveal differential gene expression in cortical bone following hindlimb unloading in female mice.

Publications

RNAseq and RNA molecular barcoding reveal differential gene expression in cortical bone following hindlimb unloading in female mice.

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