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Relationship: 2843

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Altered Signaling leads to Altered Bone Cell Homeostasis

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Altered Signaling

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Altered Bone Cell Homeostasis

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Deposition of energy leading to occurrence of bone loss	adjacent	High	Moderate	Cataia Ives (send email)	Open for citation & comment

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
human	Homo sapiens	Low	NCBI
mouse	Mus musculus	High	NCBI
rat	Rattus norvegicus	Moderate	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Male	High
Female	Low
Unspecific	High

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Adult	Moderate
Juvenile	Moderate

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Signaling pathways involved in cellular differentiation are important in the maintenance of bone cell homeostasis. This process refers to the deposition and resorption of bone matrix by osteoblasts and osteoclasts, respectively. The Wnt/ß-catenin pathway is activated in osteoblasts and the receptor activator of nuclear factor kappa Β ligand/osteoprotegerin (RANK-L/OPG) pathway regulates osteoclast differentiation. Osteoclasts originate from hematopoietic stem cells, RANK-L stimulates these progenitor cells to differentiate into pre-osteoclasts (Donaubauer et al., 2020; Smith, 2020b). Binding of RANK-L to its receptor on the osteoclast surface, RANK, triggers the expression of genes associated with osteoclastic bone resorption (Donaubauer et al., 2020). Newly formed mature osteoclasts are multi-nucleated and secrete resorptive proteins and molecules, including hydrochloric acid, tartrate-resistant acid phosphatase (TRAP), Cathepsin K (CTSK), and matrix metalloproteinase (MMP), among others, which degrade bone tissue and can be used as indicators of osteoclast activity (Smith, 2020b). As such, pathways involved in RANK-L activation are important to increased bone resorption.

Mesenchymal stem cells (MSCs) are the precursors to osteoblasts and these cells differentiate upon stimulation by signalling molecules such as tumor growth factor (TGF)-ß, Wnt, and bone morphogenic protein (BMP) (Chen, Deng and Li, 2012; Maeda et al., 2019). Alterations in these signaling pathways result in altered differentiation of MSCs and pre-osteoblasts. Early maturation of osteoblasts is regulated by runt-related transcription factor 2 (Runx2) as well as the Wnt/ß-catenin signaling pathway; altered signaling in these pathways ultimately leads to decreased production of osteoblast markers of bone deposition, including alkaline phosphatase (ALP), osteocalcin (OCN), and collagen, among others (Chatziravdeli, Katsaras and Lambrou, 2019; Manolagas and Almeida, 2007).

Tight regulation of osteoblast and osteoclast differentiation as well as bone deposition and resorption are crucial to homeostatic bone turnover. Under stress the aforementioned signaling pathways become dysregulated both internally and by external signals, resulting in altered bone cell homeostasis as measured by production of bone depositing/resorbing proteins and their by-products leading to increased osteoclast number and activity and a decrease in osteoblast number (Chatziravdeli, Katsaras and Lambrou, 2019; Donaubauer et al., 2020; Smith, 2020a; Smith, 2020b; Tian et al., 2017).

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

The strategy for collating the evidence on radiation stressors to support the relationship is described in Kozbenko et al 2022. Briefly, a scoping review methodology was used to prioritize studies based on a population, exposure, outcome, endpoint statement.

Evidence Map 2.0

ID	Experimental Design	Species	Upstream Observation	Downstream Observation	Citation (first author, year)	Notes

Evidence Map

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Title	First Author	Biological Plausibility	Dose Concordance	Temporal Concordance	Incidence Concordance

Biological Plausibility

Dose Concordance Evidence

Temporal Concordance Evidence

Incidence Concordance Evidence

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Some studies suggest radiation exposure at doses at or below 2 Gy result in no significant changes in osteoblast and osteoclast activity, as measured by ALP and TRAP expression, respectively (Kook et al., 2015; He et al., 2019). These studies, however, are inconsistent with other studies examining the effects of radiation doses from 0.25-2 Gy, which report significant, dose-dependently diminished ALP activity, and enhanced count of TRAP-positive osteoclasts (Li et al., 2020; Zhang et al., 2019). Further research is needed to elucidate the effects of lower doses of ionizing radiation on osteoblasts and osteoclasts, as well as their dose-dependent effects.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Modulating factor	Details	Effects on the KER	References
Drug	Doxycycline (autophagy inhibitor)	Treatment partially restored the radiation-induced decreases in autophagy markers as well as increased Runx2 signaling protein and ALP5 (osteoblastogenesis marker) levels.	Li et al., 2020
Drug	Anti‐HMGB1 neutralizing antibody	Treatment with 0.5 μg/ml completely prevented the increased RANK-L/OPG ratio and the increased osteoclastogenesis.	He et al., 2019
Drug	α2M	Treatment with 0.25 and 0.5 mg/mL slightly restored all endpoints of altered signaling as well as ALP activity.	Liu et al., 2018
Drug	N-acetyl cysteine (antioxidant)	Treatment reduced Nrf1 and HO-1 levels and restored Runx2 levels and ALP activity.	Kook et al., 2015
Drug	GYY4137 (25mg/kg per day)	Treatment on rats exposed to hindlimb suspension found increased levels of osteocalcin close to control levels.	Yang et al., 2019
Pulsed electromagnetic field	50 Hz, 0.6 mT pulsed electromagnetic field for 1.5 h/day during hind-limb suspension	Treatment restored signaling pathways as well as osteoblast markers to control levels.	Li et al., 2018
Drug	1 nM r-irisin	Treatment after simulated microgravity slightly restored ALP and collagen type 1 alpha-1 α1 levels.	Chen et al., 2020
Drug	DFO	Can completely inhibit osteoclast formation and bone resorption in vitro.	Zhang et al., 2019
Genetic	IL-6 knockdown	IL-6 knockdown with an IL-6 antibody partially reversed microgravity effect on all parameters of signaling pathways, osteoblastogenesis, and osteoclastogenesis	He et al., 2020

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

The following are a few examples of quantitative understanding of the relationship. All data is statistically significant unless otherwise indicated.

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Dose/Incidence Concordance

Reference	Experiment Description	Result
Li et al., 2020	In vitro. Mouse pre-osteoblastic MC3T3‑E1 was irradiated with X-rays at 0.25, 0.5, 1, 2, and 4 Gy. Runx2 transcription factor was measured to determine signaling. ALP5 activity was measured to determine osteoblastogenesis.	All endpoints changed dose-dependently. Runx2 expression and ALP5 activity both decreased a maximum of 0.4-fold after 4 Gy. Runx2 expression and ALP activity both first decreased significantly at 0.5 Gy.
Zhang et al., 2019	In vivo. 4-week-old male C57BL/6J mice were irradiated with 2 Gy X-rays at 0.23 Gy/s. Levels of NFATc1 and NF-κB transcription factors in the RANK-L/RANK pathway of osteoclastogenesis were determined. A TRAP stain was performed to determine osteoclast area.	NFATc1 increased 2.9-fold and NF-κB increased 1.5-fold after 2 Gy. TRAP-positive surface area increased 2.3-fold after 2 Gy.
He et al., 2019	In vitro. Osteocyte‐like MLO‐Y4 cells were irradiated with ¹³⁷Cs gamma rays at 2, 4, and 8 Gy. HMGB1 and the RANK-L/OPG ratio (OPG inhibits RANK-L) protein and mRNA levels were determined to measure altered signaling. Osteoclast differentiation was measured in preosteoclast RAW264.7 cells co-cultured with irradiated MLO‐Y4 cells using TRAP staining.	No significant changes were observed at 2 Gy. HMGB1 protein and mRNA levels both increased, with protein levels increasing 2.5-fold at 4 Gy and 4-fold after 8 Gy. RANK-L increased and OPG decreased shown by both protein and mRNA levels, with the RANK-L/OPG ratio of mRNA levels increasing 1.8-fold at 4 Gy and 2.5-fold at 8 Gy. The number of TRAP-positive cells increased 1.3-fold at 4 Gy and 1.8-fold at 8 Gy.
Chandra et al., 2017	In vivo. An experiment was conducted on male C57BL/6 mice (8–10 weeks) exposed to 16 Gy X-ray radiation at a rate of 1.65 Gy/min. Sclerostin, an inhibitor of the Wnt/ß-catenin pathway. Osteoblast number was determined.	16 Gy radiation exposure led to a 2.5-fold increase in sclerostin and a 0.5-fold decrease in osteoblast number.
Bai et al., 2020	In vitro. Bone marrow derived MSCs (bmMSCs), osteoblast precursors from 4-week-old male Sprague–Dawley rats were irradiated with 2, 5, and 10 Gy of ¹³⁷Cs gamma rays. The Runx2 transcription factor part of osteoblastogenic pathways was measured. ALP (osteoblastogenesis marker) activity was measured.	Runx2 decreased significantly after 2, 5, and 10 Gy, reaching a maximum 0.6-fold decrease at 10 Gy. ALP activity decreased significantly at 2, 5, and 10 Gy, following a linear trend to a maximum decrease of 48.2% (from 218 U/mg protein to 113 U/mg protein) at 10 Gy.
Liu et al., 2018	In vitro. hBMMSCs were irradiated with 8 Gy of X-rays at 1.24 Gy/min. The Runx2 transcription factor part of osteoblastogenic pathways and OGN (inhibits osteoclasts) were measured. Sox2 and Nanog (cytokine markers of stem cell pluripotency) were measured. ALP (osteoblastogenesis marker) activity was measured.	Runx2 and OGN both decreased about 0.5-fold at 8 Gy. Sox2 and Nanog both decreased more than 0.1-fold at 8 Gy. ALP activity decreased about 0.5-fold at 8 Gy.
Kook et al., 2015	In vitro. MC3T3-E1 osteoblast cells were irradiated with 2, 4, and 8 Gy of X-rays at 1.5 Gy/min. The Runx2 transcription factor mRNA levels as well as proteins in the Nrf2/HO-1 pathway were measured. ALP activity was measured to determine osteoblast function.	Runx2 mRNA decreased 0.5-fold after 8 Gy. HO-1 was increased 3-fold after 4 Gy and 5-fold after 8 Gy (non-significant increase at 2 Gy). Nrf2 increased 2.3-fold after 8 Gy. ALP activity decreased 0.3-fold after 8 Gy (non-significant decrease at 2 Gy).
Goyden et al., 2015	In vitro. The MC3T3-E1 pre-osteoblast cells were subject to microgravity. RANK-L, OPG, and sclerostin mRNA levels were measured to determine altered signaling. OCN and collagen α1 mRNA levels (osteoblast markers) were measured.	RANK-L was increased 1.3-fold, OPG decreased 0.8-fold, and sclerostin increased 1.7-fold. OCN and collagen α1 were decreased 0.6-fold.
He et al., 2020	In vivo and in vitro. Male 10-week-old C57BL/6J mice were subject to hind-limb suspension. MC3T3-E1 cells were exposed to modeled microgravity. The RANK-L/OPG ratio of signaling molecules was determined. ALP and OCN for osteoblasts and TRAP for osteoclasts were determined.	In the hind-limb suspended mice, RANK-L/OPG ratio increased 3.5-fold, ALP decreased 0.3-fold, OCN decreased 0.5-fold, TRAP increased 2-fold. In MC3T3-E1 cells, RANK-L expression was increased 75% and OPG decreased 33%. This was accompanied by a ~50% in ALP mRNA expression and a 0.4-fold decrease in ALP activity.
Li et al., 2018	In vivo. Female 3-month-old Wistar rats were subjected to microgravity for 4 weeks. Runx2, OSX, BMP-2, RANK-L, OPG signaling proteins and components of the sAC/cAMP/PKA/CREB signaling pathway were measured. OCN and PIPN were measured for osteoblastogenesis and TRAP5b and CTX-1 were measured for osteoclastogenesis in serum.	Runx2 decreased 0.3-fold, OSX 0.4-fold, BMP-2 0.1-fold, OPG/RANK-L 0.2-fold. Phosphorylated PKA and CREB both decreased more than 0.5-fold. Osteoblast markers decreased about 0.5-fold, while osteoclast markers increased about 1.5-fold.
Rucci et al., 2007	In vitro. Calvaria and primary osteoclasts from 7-day-old CD1 mice were differentiated into osteoblasts and osteoclasts, respectively, and exposed to microgravity at 0.08 G or 0.008 G for 24 h. The RANK-L/OPG ratio was determined. ALP activity (osteoblast marker) and TRAP level (osteoclast marker) were determined.	The RANK-L/OPG ratio showed a nonsignificant 1.4-fold increase after 0.08 G and a 4-fold increase after 0.008 G. TRAP increased 2.4-fold after 0.08 G and 5.6-fold after 0.008 G. ALP activity and expression did not significantly change.
Saxena et al., 2011	In vitro. RAW264.7 murine macrophage cells and mouse bone marrow macrophage precursors were exposed to microgravity. All cells were cultured with RANK-L. The signaling molecules ERK, p38, NFATc1, and PLCγ2 were measuredt. TRAP and CTSK mRNA levels (osteoclast markers) were measured.	Phosphorylated ERK, PLCγ2, and p38 as well as NFATc1 were increased after microgravity. TRAP and CTSK increased 3.5-fold in RAW264.7 cells. TRAP increased 3-fold and CTSK increased 7.5-fold in mouse bone marrow macrophages.
Yang et al., 2019	In vivo and in vitro. Rats were exposed to microgravity conditions by hindlimb suspension. An in vitro model used MC3T3-E1 (osteoblast-like cells) in a bone cell differentiation media exposed to microgravity conditions. RANK-L and OPG were measured as part of RANK signaling pathway. Plasma H2S concentration, a gasotransmitter serving many physiological/pathophysiological roles, and endogenous H2S produced by osteoblasts were monitored. Osteoblastogenesis was measured by serum OCN and ALP.	Concentration of RANK-L increased significantly by 1.5–fold, while OPG concentration decreased by 0.71–fold. Endogenous H2S production by osteoblasts and concentration in plasma were decreased 0.66-fold. ALP activity decreased 0.53-fold after microgravity simulation in rats. OCN levels in sera of rats exposed to hindlimb suspension decreased 0.6-fold. Rats experienced a 3-fold increase in tibia IL-6, while osteoblasts supernatant had a 4-fold increase in IL-6.
Chen et al., 2020	In vivo and in vitro. 2-month-old mice were subject to hindlimb unloading to simulate microgravity. An in vitro model of primary osteoblasts isolated from murine femurs were exposed to microgravity for 48-hours. β-catenin mRNA and protein expression were determined. ALP, an osteoblast marker, and collagen type 1 alpha-1 were measured as osteoblastogenesis markers.	Following hindlimb unloading, PCR analysis of β-catenin showed decreased expression by 0.45-fold in both in vivo mice after 28 days and in vitro primary osteoblasts after 48 h. In vitro β-catenin protein expression decreased by 0.5-fold. The mRNA expressions of ALP and collagen type 1 alpha-1 were downregulated by 93.9% and 62.4%, respectively, in vivo, and were both downregulated by 60% in vitro.
Sambandam et al., 2016	In vitro. Osteoclast cells were taken from the bone marrow of 6- to 8-week-old C57BL/6 mice and exposed to 0.008 G for 24h. The mRNA of TRAF6 signaling molecule downstream of RANK was measured. The mRNA of TRAIL (proliferative signaling molecule) was also measured. TRAP staining was performed to measure osteoclastogenesis. Western blots were also performed to confirm changes in mRNA levels.	Following 0.008G, signaling molecules TRAF6 and TRAIL increased 6-fold and 14.5-fold, respectively. TRAP increased 1.7-fold after 0.008G.

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Time Concordance

Reference	Experiment Description	Result
Li et al., 2020	In vitro. Mouse pre-osteoblastic MC3T3‑E1 was irradiated with X-rays at various doses. Runx2 transcription factor was measured to determine signaling. ALP5 activity was measured to determine osteoblastogenesis.	Runx2 and ALP5 activity both decreased a maximum of 0.4-fold after 72 h. ALP5 activity was also observed decreased the same amount after 1 and 2 weeks.
Zhang et al., 2019	In vivo. 4-week-old male C57BL/6J mice were irradiated with 2 Gy X-rays at 0.23 Gy/s. Levels of NFATc1 and NF-κB transcription factors in the RANK-L/RANK pathway of osteoclastogenesis were determined. A TRAP stain was performed to determine osteoclast area.	NFATc1 increased 2.9-fold and NF-κB increased 1.5-fold after 28 days. TRAP-positive surface area increased 2.3-fold after 28 days.
Liu et al., 2018	In vitro. hBMMSCs were irradiated with 8 Gy of X-rays at 1.24 Gy/min. The Runx2 transcription factor was measured. Sox2 and Nanog (cytokine markers of stem cell pluripotency) were measured. ALP (osteoblastogenesis marker) activity was measured.	Sox2 and Nanog both decreased more than 0.1-fold after 24h. Runx2 decreased about 0.5-fold at 1 week. ALP activity decreased about 0.5-fold at 1 week.
Kook et al., 2015	In vitro. MC3T3-E1 osteoblast cells were irradiated with X-rays at 1.5 Gy/min. The mRNA of Runx2 transcription factor well as proteins in the Nrf2/HO-1 pathway were measured. ALP activity and mRNA level were measured to determine osteoblast function.	Runx2 mRNA decreased 0.5-fold at 1-3 days after 8 Gy irradiation. HO-1 was increased 4.5-fold at 2 days. Nrf2 increased 2.3-fold at 1 day. ALP activity decreased 0.3-fold after 7 days.
Goyden et al., 2015	In vitro. The MC3T3-E1 pre-osteoblast cells were subject to microgravity at 0 G. The mRNA of RANK-L, OPG, and sclerostin was measured to determine altered signaling. The mRNA of OCN and collagen α1 (osteoblast markers) was measured to determine osteoblast function.	RANK-L was increased 1.3-fold, OPG decreased 0.8-fold, and sclerostin increased 1.7-fold after 48 h of microgravity. OCN and collagen α1 were decreased 0.6-fold after 48 h of microgravity. IL-6 increased 2-fold after 48 h, where the maximum change in OCN was observed, but not after 12 h.

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Not Identified

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

The evidence for the taxonomic applicability to humans is low as majority of the evidence is from in vitro human-derived cells and in vivo animal models. The relationship is supported primarily by studies from mice models and rat models. The relationship has been shown in both male and female animal models and plausible at any life stage. However, majority of studies use preadolescence and adolescence animal models.