TY - JOUR
T1 - Gene expression of intracortical bone demonstrates loading-induced increases in Wnt1 and Ngf and inhibition of bone remodeling processes
AU - Harris, Taylor L.
AU - Silva, Matthew J.
N1 - Funding Information:
This work was supported by NIH grants R01AR047867 and T32EB018266 and the Washington University Musculoskeletal Research Center (P30AR074992). We acknowledge the Developmental Biology Microscopy Core at Washington University in St. Louis for use of the laser capture microscope. We thank Crystal Idleburg and Samantha Coleman for assistance with histology. We thank the Genome Technology Access Center (supported by P30CA91842 and UL1TR002345), especially Twyla Juehne and Mala Sharma for their important role in designing and implementing the processing of the RNA for hybridization to microarray probes and Jinsheng Yu for analysis of raw intensity values obtained from microarray chips. We also acknowledge NIH shared instrumentation grant S10 RR0227552 for use of the Nanozoomer 2.0HT microscope. We additionally thank Dr. Farshid Guilak for use of the RNAscope hybridization oven for the in situ assay. For help with protocols for RNAscope we thank Dr. Marc Wein and for MMP13 IHC we thank Dr. Tamara Alliston.
Funding Information:
This work was supported by NIH grants R01AR047867 and T32EB018266 and the Washington University Musculoskeletal Research Center ( P30AR074992 ). We acknowledge the Developmental Biology Microscopy Core at Washington University in St. Louis for use of the laser capture microscope. We thank Crystal Idleburg and Samantha Coleman for assistance with histology. We thank the Genome Technology Access Center (supported by P30CA91842 and UL1TR002345), especially Twyla Juehne and Mala Sharma for their important role in designing and implementing the processing of the RNA for hybridization to microarray probes and Jinsheng Yu for analysis of raw intensity values obtained from microarray chips. We also acknowledge NIH shared instrumentation grant S10 RR0227552 for use of the Nanozoomer 2.0HT microscope. We additionally thank Dr. Farshid Guilak for use of the RNAscope hybridization oven for the in situ assay. For help with protocols for RNAscope we thank Dr. Marc Wein and for MMP13 IHC we thank Dr. Tamara Alliston.
Publisher Copyright:
© 2021 Elsevier Inc.
PY - 2021/9
Y1 - 2021/9
N2 - Osteocytes are the primary mechanosensitive cells in bone. However, their location in mineralized matrix has limited the in vivo study of osteocytic genes induced by mechanical loading. Laser Capture Microdissection (LCM) allows isolation of intracortical bone (Intra-CB), enriched for osteocytes, from bone tissue for gene expression analysis. We used microarray to analyze gene expression from mouse tibial Intra-CB dissected using LCM 4 h after a single loading bout or after 5 days of loading. Osteocyte enrichment was supported by greater expression of Sost, Dmp1, Dkk1, and Mepe in Intra-CB regions vs. Mixed regions containing periosteum and muscle (fold-change (FC) = 3.4, 2.2, 5.1, 3.0, respectively). Over 150 differentially expressed genes (DEGs) due to loading (loaded vs. contralateral control) in Intra-CB were found on Day 1 and Day 5, but only 10 genes were differentially expressed on both days, including Ngf (Day 1 FC = 13.5, Day 5 FC = 11.1) and Wnt1 (Day 1 FC = 1.5, Day 5 FC = 5.1). The expression of Ngf and Wnt1 within Intra-CB was confirmed by in situ hybridization, and a significant increase in number of Wnt1 mRNA molecules occurred on day 1. We also found changes in extracellular matrix remodeling with Timp1 (FC = 3.1) increased on day 1 and MMP13 (FC = 0.3) decreased on day 5. Supporting this result, IHC for osteocytic MMP13 demonstrated a marginal decrease due to loading on day 5. Gene Ontology (GO) biological processes for loading DEGs indicated regulation of vasculature, neuronal and immune processes while cell-type specific gene lists suggested regulation of osteoclast, osteoblast, and endothelial related genes. In summary, microarray analysis of microdissected Intra-CB revealed differential regulation of Ngf, Wnt1, and MMP13 due to loading in osteocytes.
AB - Osteocytes are the primary mechanosensitive cells in bone. However, their location in mineralized matrix has limited the in vivo study of osteocytic genes induced by mechanical loading. Laser Capture Microdissection (LCM) allows isolation of intracortical bone (Intra-CB), enriched for osteocytes, from bone tissue for gene expression analysis. We used microarray to analyze gene expression from mouse tibial Intra-CB dissected using LCM 4 h after a single loading bout or after 5 days of loading. Osteocyte enrichment was supported by greater expression of Sost, Dmp1, Dkk1, and Mepe in Intra-CB regions vs. Mixed regions containing periosteum and muscle (fold-change (FC) = 3.4, 2.2, 5.1, 3.0, respectively). Over 150 differentially expressed genes (DEGs) due to loading (loaded vs. contralateral control) in Intra-CB were found on Day 1 and Day 5, but only 10 genes were differentially expressed on both days, including Ngf (Day 1 FC = 13.5, Day 5 FC = 11.1) and Wnt1 (Day 1 FC = 1.5, Day 5 FC = 5.1). The expression of Ngf and Wnt1 within Intra-CB was confirmed by in situ hybridization, and a significant increase in number of Wnt1 mRNA molecules occurred on day 1. We also found changes in extracellular matrix remodeling with Timp1 (FC = 3.1) increased on day 1 and MMP13 (FC = 0.3) decreased on day 5. Supporting this result, IHC for osteocytic MMP13 demonstrated a marginal decrease due to loading on day 5. Gene Ontology (GO) biological processes for loading DEGs indicated regulation of vasculature, neuronal and immune processes while cell-type specific gene lists suggested regulation of osteoclast, osteoblast, and endothelial related genes. In summary, microarray analysis of microdissected Intra-CB revealed differential regulation of Ngf, Wnt1, and MMP13 due to loading in osteocytes.
KW - Bone formation
KW - Gene expression
KW - Laser Capture Microdissection
KW - Mechanical loading
KW - Osteocytes
UR - http://www.scopus.com/inward/record.url?scp=85107033168&partnerID=8YFLogxK
U2 - 10.1016/j.bone.2021.116019
DO - 10.1016/j.bone.2021.116019
M3 - Article
C2 - 34023542
AN - SCOPUS:85107033168
SN - 8756-3282
VL - 150
JO - Bone
JF - Bone
M1 - 116019
ER -