Transcriptional Profiling of Laser Capture Microdissected Subpopulations of the Osteoblast Lineage Provides Insight Into the Early Response to Sclerostin Antibody in Rats

Abstract

Sclerostin antibody (Scl-Ab) increases bone formation through a process dependent on the activation of canonical Wnt signaling, although the specific signaling in the osteoblast lineage in vivo is largely unknown. To gain insight into the signaling pathways acutely modulated by Scl-Ab, the transcriptional response of subpopulations of the osteoblast lineage was assessed by TaqMan and microarray analyses of mRNA isolated from laser capture microdissection (LCM)-enriched samples from the vertebrae of ovariectomized rats during the first week after Scl-Ab administration. Briefly, 6-month-old Sprague-Dawley rats were ovariectomized and, after 2 months, received a single dose of vehicle (VEH) or 100 mg/kg Scl-Ab (n ¼ 20/group). Lumbar vertebrae were collected at 6, 24, 72, and 168 hours postdose and cryosectioned for LCM. Osteocytes were captured from bone matrix, and osteoblasts and lining cells were captured from bone surfaces based on fluorochrome labeling. mRNA was isolated, amplified, and profiled by TaqMan and microarray. Expression analysis revealed that Scl-Ab caused strikingly similar transcriptional profiles across all three cell types. Only 13 known canonical Wnt target genes, the majority with known functions in bone, showed a significant change in expression by microarray in response to Scl-Ab, with Wisp1 and Twist1 being the most responsive. Coincident with increased expression of Wnt target genes was the upregulation of numerous extracellular matrix (ECM) genes. The acute and progressive upregulation of ECM genes in lining cells supports their activation into matrix-producing osteoblasts, consistent with modelingbased bone formation. A similar transcriptional profile in osteocytes may indicate that Scl-Ab stimulates perilacunar/pericanalicular matrix deposition. Pathway analyses indicated that Scl-Ab regulated a limited number of genes related to cell cycle arrest and B-cell development. These data describe the acute downstream signaling in response to Scl-Ab in vivo and demonstrate selected canonical Wnt target gene activation associated with increased bone formation in all mature osteoblast subpopulations.

Old age and the associated impairment of bones' adaptation to loading are associated with transcriptomic changes in cellular metabolism, cell-matrix interactions and the cell cycle

“…Given that only small numbers of periosteal cells are present on the surface of the cortical shells used in our analysis and that the predominant cell type present is the terminally differentiated osteocyte (Kelly et al, 2014), the robust proliferation-related signature was unexpected. However, consistent with our observation, activation of Wnt signalling with anti-sclerostin antibodies has previously been reported to alter expression of proliferation-related genes in osteocyte-rich samples (Nioi et al, 2015, Taylor et al, 2016). One explanation for this proliferation-related signature is that it reflects changes in osteocyte senescence and predisposition to apoptosis, with apoptosis-related terms being significantly enriched in the loading-responsive gene signature from the aged mice as well as in the list of genes differentially expressed between young and aged.…”

The Osteocyte Transcriptome Is Extensively Dysregulated in Mouse Models of Osteogenesis Imperfecta

“…Although it is known that several aspects of the transcriptome and proteome change during the osteoblast‐to‐osteocyte transition, Col1a1 and Col1a2 are among the most copious transcripts in both cell types. This observation confirms previous findings from others, showing that type I collagen expression levels are very similar in osteoblasts and osteocytes and that Col1a1 is expressed at even higher levels in osteocytes compared with osteoblasts during the early anabolic response to a sclerostin antibody in a rat model . We show here that type I collagen expression is significantly upregulated in both mouse models of OI (together with Bglap and Sparc ), suggesting perhaps a futile attempt to increase bone formation.…”

“…This observation confirms previous findings from others, showing that type I collagen expression levels are very similar in osteoblasts and osteocytes (39) and that Col1a1 is expressed at even higher levels in osteocytes compared with osteoblasts during the early anabolic response to a sclerostin antibody in a rat model. (52) We show here that type I collagen expression is significantly upregulated in both mouse models of OI (together with Bglap and Sparc), suggesting perhaps a futile attempt to increase bone formation. ER stress responses to misfolded procollagen chains can contribute to the pathogenesis of OI mouse models.…”

Estrogen Regulates Bone Turnover by Targeting RANKL Expression in Bone Lining Cells

“…We used these high quality cryosections of undecalcified bone for laser capture microdissection, permitting sensitive in situ mRNA profiling of osteoblasts, osteocytes, and bone lining cells. In contrast to a recently published and independently developed LCM method based on labeling of bone forming surfaces by in vivo fluorochrome labeling in rats 58 , our method is based on morphological criteria to define the different cell types. Besides species differences, these methodological differences may explain the discrepancies between the cell type-specific mRNA expression profiles yielded by the different technologies.…”