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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	Vinita Chauhan (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 Supporting this KER

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

Overall weight of evidence: High

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

The biological rationale for linking altered signaling pathways to altered bone cell homeostasis is strongly supported by a number of review articles published on the subject. A recent review by Donaubauer et al. (2020) discusses internal and external signaling pathways in osteoblasts and osteoclast that are influenced from exposure to a multitude of stressors. A number of reviews also discuss signaling pathways affecting osteoblast and osteoclast differentiation as well as the integral role osteoblasts play in the differentiation of osteoclasts through the RANK-L/OPG pathway (Arfat et al., 2014; Bellido, 2014; Boyce and Xing, 2007; Chatziravdeli, Katsaras and Lambrou, 2019; Chen, Deng and Li, 2012; Donaubauer et al., 2020; Maeda et al., 2019; Manolagas and Almeida, 2007; Smith, 2020a; Smith, 2020b; Willey et al., 2011).

The RANK/RANK-L pathway plays a central role in the differentiation of osteoclasts, as both RANK-L and OPG, an inhibitor of RANK-L, are secreted by osteoblasts and osteocytes (Boyce and Xing, 2007; Donaubauer et al., 2020). The upregulation of RANK-L and downregulation of OPG secretion by osteoblasts indirectly affect osteoclasts and ultimately increase the resorption of bone matrix (Chatziravdeli, Katsaras and Lambrou, 2019; Donaubauer et al., 2020).

RANK-L, upon binding to its receptor on the osteoclast surface, RANK, internally activates cytokine NF-kB in osteoclasts, as well as growth and survival signaling cascades of extracellular signal-regulated kinase (ERK), TNF, and IL-6, preventing apoptosis and promoting differentiation of osteoclasts (Donaubauer et al., 2020; Tian et al., 2017). Over-expression of RANK-L will over-stimulate these downstream pathways leading to the activation of the master transcription factor of osteoclasts, nuclear factor of activated T cells 1 (NFATc1). NFATc1 is responsible for the transcription of genes specific to osteoclastic bone resorption including TRAP and CTSK (Donaubauer et al., 2020; Smith, 2020b). Over expression of RANK-L results in increased transcription of TRAP and CTSK genes and ultimately, increased bone resorption.

Osteoblastogenesis itself is also tightly regulated by external signals, of which Wnt (activator of Wnt/ß-catenin pathway) is often discussed in the literature (Arfat et al., 2014; Chen, Deng and Li, 2012; Maeda et al., 2019; Smith, 2020b). The canonical Wnt/ß-catenin pathway plays a central role in osteoblast differentiation, as Wnt stimulation preserves ß-catenin from ubiquitination/ degradation, allowing it to translocate to the nucleus and induce expression of key osteoblast genes (Maeda et al., 2019; Manolagas and Almeida, 2007). Dysregulation of key components in this pathway result in significantly depressed protein expression/activity of ALP and OCN, implicating this pathway in the depression of osteoblastic bone deposition (Arfat et al., 2014; Maeda et al., 2019; Manolagas and Almeida, 2007; Tian et al., 2017). As such, Wnt signaling is of paramount importance for preservation of bone mass, as ß-catenin commits precursors to the osteoblast lineage (Manolagas and Almeida, 2007; Tian et al., 2017). Runx2 and Osterix (OSX), among others, are also key transcription factors involved in the early maturation osteoblasts, as they advance the progressive differentiation of MSCs and coordinate the expression of key proteins essential to osteoblast function; downregulation of Runx2 and OSX in osteoblasts is concordant with decreases in ALP and OCN activity (Arfat et al., 2014; Chatziravdeli, Katsaras and Lambrou, 2019).

Although less direct, altered osteocyte signaling also plays a key role in the loss of homeostasis among bone cells as osteocytes are the most abundant cell type in bones and are key regulators of bone metabolism. Osteocytes can stimulate osteoclastogenesis by increasing production and release of high mobility group box 1 (HMGB1) and elevating the RANK-L/OPG ratio, inducing the maturation of osteoclast precursors and promoting bone resorption (Arfat et al., 2014; Donaubauer et al., 2020; He et al., 2019). Further, osteocytes with increased expression of Dkk1 and sclerostin result in potent antagonization of bone morphogenic proteins (BMPs) and diversion of LRP5/6 (coreceptors in the Wnt pathway) from Wnt signaling, ultimately inhibiting osteoblast differentiation (Bellido, 2014; Chandra et al., 2017).

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

The empirical data obtained for this KER strongly supports a link of altered signaling pathways leading to altered bone cell homeostasis. The majority of empirical evidence is derived from research using various stressors including X-rays and gamma rays as well as microgravity. These exposures are both known to directly/indirectly induce alterations in relevant signaling pathways of bone cells leading to the deposition and resorption of bone in a dose-dependent manner (Bai et al., 2020; Chandra et al., 2017; Chen et al., 2020; Goyden et al., 2015; He et al., 2019; He et al., 2020; Kook et al., 2015; Li et al., 2020; Liu et al., 2018; Rucci et al., 2007; Sambandam et al., 2016; Saxena et al., 2011; Yang et al., 2019; Zhang et al., 2019).

Incidence concordance

There is some evidence that signaling pathways demonstrate greater changes following a stressor than altered bone cell homeostasis. He et al. (2019) demonstrated this in osteocytes irradiated with 4 and 8 Gy of gamma rays through increases to HMGB1 and the RANK-L/OPG ratio that were greater than the increases to osteoclast numbers. X-ray irradiation of mice at 16 Gy resulted in a 2.5-fold increase in sclerostin (Wnt/β-catenin pathway inhibitor) and a 0.5-fold decrease in osteoblast number (Chandra et al., 2017). After 8 Gy of X-ray irradiation of human bone marrow mesenchymal stem cells (hBMMSCs) Sox2 and Nanog decreased to less than 0.1-fold, while ALP activity decreased 0.5-fold (Liu et al., 2018). Microgravity exposure to mice increased the RANK-L/OPG ratio 3.5-fold while osteoblast markers decreased a maximum of 0.3-fold and osteoclast markers increased a maximum of 2-fold (He et al., 2020). Microgravity exposure to rats also led to decreases in osteoblast signaling molecules between 0.4- and 0.1-fold and a 5-fold increase in the RANK-L/OPG ratio (Li et al., 2018). This led to a 0.5-fold decrease in osteoblast markers and a 1.5-fold increase in osteoclast markers (Li et al., 2018). Also under microgravity, osteoclast cells showed 6-fold increased TRAF6 and 14.5-fold increased TRAIL, while the osteoclast marker TRAP increased 1.7-fold (Sambandam et al., 2016).

Dose Concordance

Strong evidence exists in the current literature suggesting a dose concordance between alterations of signaling pathways and altered bone cell homeostasis. Exposure to radiation (X-rays and gamma rays) ranging from 0.25-12 Gy and microgravity in mice, rat, and osteoblast cell models shows significant linear dose-dependent diminishment of signaling molecules essential to osteoblast differentiation, including Runx2, Sox2/Nanog, H₂S and ß-catenin. Studies observing diminishment of these signaling molecules present significant dose-dependent linear decreases in ALP and OCN activity/expression as well, indicating depressed osteoblast function as a result (Bai et al., 2020; Chen et al., 2020; Li et al., 2020; Liu et al., 2018). Further signaling changes in osteoblasts occur under low-to-high dose radiation (0.25 to >2 Gy) and microgravity, with significant increases in osteoblast production of sclerostin, inhibitor of the Wnt/ß-catenin pathway. These changes result in significant linear dose-dependent decreases in ALP activity and osteoblast number at radiation doses greater than 0.25 Gy and/or microgravity exposure (Chandra et al., 2017; Goyden et al., 2015; Li et al., 2020).

One study showed dysregulation of the nuclear factor erythroid 2-related factor/ heme oxygenase-1 (Nrf2/HO-1) pathway and downstream effects on bone metabolism. Dose-dependent increases in protein expression of both Nrf2 and HO-1 were observed following high doses of radiation exposure (>2 Gy) with linear dose-dependent decreases in ALP activity in osteoblasts (Kook et al., 2015). Another study examined hydrogen sulfide level, a known gasotransmitter (a class of neurotransmitters) serving many physiological and pathophysiological functions. Decreased levels of this transmitter by microgravity exposure similarly reduced OCN activity and ALP expression in osteoblasts (Yang et al., 2019).

Osteoblasts and osteocytes have also been shown to upregulate the production of cytokines (interleukin (IL)-6 and RANK-L, an osteoclastogenic cytokine) following low and high doses of radiation or microgravity exposure. Alterations in these signalling molecules resulted in upregulation in bone resorption and expression of TRAP (He et al., 2019; He et al., 2020; Rucci et al., 2007). Further, production of OPG, a RANK-L inhibitor, by osteoblasts is significantly diminished under radiation and microgravity exposure, strengthening the stimulatory effect of RANK-L on osteoclasts leading to enhanced expression of TRAP and bone resorption pit area (He et al., 2019; He et al., 2020; Rucci et al., 2007; Yang et al., 2019).

Osteocytes irradiated with gamma rays >2 Gy showed significant linear dose-dependent upregulation in HMGB1, a signalling molecule released by apoptotic osteocytes involved in osteoclast recruitment. Upregulation of HMGB1 resulted in a similar dose-dependent increase in osteoclast count, along with upregulation in the RANK-L/OPG ratio indicating increased resorptive activity (He et al., 2019). Osteoclastogenesis pathways downstream to RANK-L-induced activation also show significant dysregulation under microgravity or ionizing radiation exposure. Microgravity exposure resulted in the upregulation of tumor necrosis factor receptor-associated factor 6 (TRAF6), an osteoclastogenic signaling molecule activated by RANK, and tumor necrosis factor-related apoptosis inducing ligand (TRAIL), an inhibitor of OPG, resulting in significantly enhanced osteoclast count and osteoclastogenesis (Sambandam et al., 2016). Osteoclasts exposed to microgravity, or 2 Gy X-rays show significant upregulation in NFATc1, the master transcription factor for osteoclastogenesis, and nuclear factor kappa B (NF-kB), an inducible cytokine transcription factor. Upregulation of NFATc1 and NF-kB results in severely enhanced TRAP expression, osteoclast area, and resorption pit area, indicating increased bone resorption (Saxena et al., 2011; Zhang et al., 2019). Further, phosphorylation of intracellular signaling components, ERK and phospholipase C (PLCγ2), involved in cell survival and proliferation are upregulated in microgravity-exposed osteoclasts. Enhanced ERK and PLCγ2 results in an enhanced count of TRAP-positive multinucleated osteoclasts, indicating increased bone resorption (Saxena et al., 2011).

With the exception of the study by Rucci et al. (2007), studies that examined the effects of a range of doses of radiation on a single model found that significant changes to signaling pathways occurred at lower or equal doses than increases in altered bone cell homeostasis, thus providing evidence for dose concordance between the upstream and downstream KEs (Bai et al., 2020; He et al., 2019; Kook et al., 2015; Li et al., 2020). For example, Bai et al. (2020) showed in vitro that both signaling molecule Runx2 and osteoblast activity significantly decreased at all doses from 2-10 Gy of gamma irradiation. Similarly, osteocytes irradiated with gamma rays showed changes in the expression of multiple signaling molecules after 4 and 8 Gy but not after 2 Gy, while TRAP-positive osteoclasts increased at 4 and 8 Gy as well, but also not after 2 Gy (He et al., 2019). Kook et al. (2015) used X-rays at the same doses and found altered signaling at 4 and 8 Gy but not at 2 Gy. Osteoblast activity decreased at 4 and 8 Gy, but not at 2 Gy (Kook et al., 2015). Using slightly lower doses, Li et al. (2020) found that altered expression of signaling molecule Runx2 and decreased osteoblast activity both occurred at the same dose of 0.5 Gy, but neither changed at 0.25 Gy.

Time Concordance

Many studies using in vitro mouse and human as well as in vivo mouse models exposed to microgravity and X-ray irradiation from 2 to 8 Gy show that bone cell altered bone cell homeostasis occurs at the same time or after altered signaling in a time-course. Altered signaling molecules including Runx2, RANK-L, OPG and Nrf2 were mostly found altered 1 to 3 days after a stressor (Goyden et al., 2015; Kook et al., 2015; Li et al., 2020; Liu et al., 2018). Bone cell markers were frequently found decreased weeks after a stressor (Kook et al., 2015; Li et al., 2020; Liu et al., 2018; Zhang et al., 2019).

Essentiality

Studies examining the inhibition or knock-down of signaling molecules strongly support the relationship between altered signaling pathways and bone cell altered bone cell homeostasis. In one study, treatment with OPG, an inhibitor for RANK-L, reversed the effect of microgravity on osteoclast activity, decreasing it to well-below control levels, suggesting a role for RANK-L in microgravity-induced osteoclastogenesis (Rucci et al., 2007). Treatment with doxycycline, known to inhibit autophagy in osteoclasts, reversed the effect of irradiation on the osteoblastogenic transcription factor Runx2, ultimately restoring ALP activity at X-ray doses of 0.25-4 Gy completely to control levels (Li et al., 2020).

Sclerostin is a protein known to inhibit the Wnt/ß-catenin canonical pathway by competing for the Wnt receptor. Chandra et al. (2017) observed that sclerostin knock-out increased osteoblast activity and decreased osteoclast activity, by replenishing ß-catenin protein expression, thereby strongly favouring osteoblastogenesis. Further, overexpression of ß-catenin in osteoblasts has been shown to reverse the effect of simulated microgravity on ß-catenin protein expression, and partially reversing its effect on ALP staining area; ß-catenin knockdown had the opposite effect under microgravity (Chen et al., 2020). Knockdown of TRAIL, which induces osteoclastogenesis by sequestering the RANK-L inhibitor OPG, reversed the effect of microgravity on osteoclast numbers (Sambandam et al., 2016). HMGB1 is a protein released by apoptotic osteocytes that mediates RANK-L-induced osteoclastogenesis by interacting with receptor for advanced glycation end-products (RAGE); He and associates confirmed this under gamma radiation-induced osteoclastogenesis, as treatment with HMGB1 antibody fully reversed the effect of radiation on osteoclast count (He et al., 2019; Zhou et al., 2008). A role for hydrogen sulfide in osteoblast and osteoclast differentiation under microgravity has also been suggested, as treatment with H2S donor GYY4137 leads to decreased RANK-L/OPG production ratio by osteoblasts and increased ALP activity (Yang et al., 2019).

α-macroglobulin (α2M) is a glycoprotein known to exert radioprotective effects on cells, and treatment of osteoblasts with α2M was shown to significantly reverse the effect of radiation on protein expression of transcription factors Runx2 and Sox2, and osteoglycin (OGN), while also reversing its effect on ALP activity, returning the values to control levels (Liu et al., 2018). A significant role for iron in the induction of osteoclastogenesis under both radiation and non-radiation conditions was posited, as treatment with iron chelator deferoxamine mesylate (DFO) fully decreased serum ferritin and iron levels, while also decreasing osteoclast and resorption pit area by 100% in both irradiated and non-irradiated groups (Zhang et al., 2019).

Exposure to pulsed electromagnetic fields (PEMFs) has also shown promise in improving the effects of modeled microgravity on measures of bone cell function. In one study, PEMF exposure together with hindlimb suspension of rats showed significant improvement in protein expression related to bone cell function, with increased expression of Runx2 and OSX (involved in early osteoblast maturation), and BMP-2 (an osteoblast stimulatory molecule), along with significant decrease in the RANK-L/OPG ratio (osteoclast stimulatory molecule and its inhibitor) relative to the hindlimb suspension alone group. A role of the sAC/cAMP/PKA/CREB signaling pathway was also implicated in these improvements, as phosphorylation of its key components, including protein kinase A (PKA) and (cAMP response element-binding protein) CREB, and expression of soluble adenylyl cyclases (sAC) and cAMP were significantly improved in comparison to the hindlimb suspended group. These changes were ultimately accompanied by significant improvements in bone deposition markers osteocalcin and propeptide of type I procollagen (PIPN) and decreases in bone resorption markers TRAP5b and collagen C-terminal telopeptide (CTX)-1 (Li et al., 2018).

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.

References

List of the literature that was cited for this KER description. More help

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