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Background
The bone marrow niche supports hematopoietic cell development through intimate contact with multipotent stromal mesenchymal stem cells; however, the intracellular signaling, function, and regulation of such supportive niche cells are still being defined. Our study was designed to understand how G protein receptor kinase 3 (GRK3) affects bone marrow mesenchymal stem cell function by examining primary cells from GRK3-deficient mice, which we have previously published to have a hypercellular bone marrow and leukocytosis through negative regulation of CXCL12/CXCR4 signaling.
Methods
Murine GRK3-deficient bone marrow mesenchymal stromal cells were harvested and cultured to differentiate into three lineages (adipocyte, chondrocyte, and osteoblast) to confirm multi-potency and compared to wild type cells. Immunoblotting, modified-TANGO experiments, and flow cytometry were used to further examine the effects of GRK3 deficiency on bone marrow mesenchymal stromal cell receptor signaling. Microcomputed tomography was used to determine trabecular and cortical bone composition of GRK3-deficient mice and standard ELISA to quantitate CXCL12 production from cellular cultures.
Results
GRK3-deficient, bone marrow-derived mesenchymal stem cells exhibit enhanced and earlier osteogenic differentiation in vitro. The addition of a sphingosine kinase inhibitor abrogated the osteogenic proliferation and differentiation, suggesting sphingosine-1-phosphate receptor signaling was a putative G-protein coupled receptor regulated by GRK3. Immunoblotting showed prolonged ERK1/2 signaling after stimulation with sphingosine-1-phosphate in GRK3-deficient cells, and modified-TANGO assays suggested the involvement of β-arrestin-2 in sphingosine 1 phosphate receptor internalization.
Conclusions
Our work suggests that GRK3 regulates sphingosine-1-phosphate receptor signaling on bone marrow mesenchymal stem cells by recruiting β-arrestin to the occupied GPCR to promote internalization, and lack of such regulation affects mesenchymal stem cell functionality.
Keywords
Bone marrow-derived mesenchymal stem cells, mesenchymal stem cells, G protein-coupled receptors (GPCRs), G protein-coupled receptor kinase 3 (GRK3), Sphingosine-1-phosphate (S1P), Sphingosine-1-phosphate receptor (S1PR), Osteogenic differentiation, Proliferation
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CURRENT STATUS: Published
View the published article at Stem Cell Research & Therapy.
Version 1
Posted 18 Mar, 2021
No community comments so far
Editorial decision: Minor Revision
On 23 Apr, 2021
Review #2 received
Received 14 Apr, 2021
Review #3 received
Received 14 Apr, 2021
Review #1 received
Received 12 Apr, 2021
Reviewer #3 agreed
On 06 Apr, 2021
Reviewer #2 agreed
On 06 Apr, 2021
Reviewer #1 agreed
On 04 Apr, 2021
Reviews received
Received 04 Apr, 2021
Reviewers invited
Invitations sent on 23 Mar, 2021
Submission checks complete
On 13 Mar, 2021
Editor invited
On 13 Mar, 2021
Editor assigned
On 08 Mar, 2021
First submitted
On 08 Mar, 2021
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A new preprint design is coming!
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See the published version of this preprint at Stem Cell Research & Therapy.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Stem Cell Research & Therapy.
Version 1
Posted 18 Mar, 2021
No community comments so far
Editorial decision: Minor Revision
On 23 Apr, 2021
Review #2 received
Received 14 Apr, 2021
Review #3 received
Received 14 Apr, 2021
Review #1 received
Received 12 Apr, 2021
Reviewer #3 agreed
On 06 Apr, 2021
Reviewer #2 agreed
On 06 Apr, 2021
Reviewer #1 agreed
On 04 Apr, 2021
Reviews received
Received 04 Apr, 2021
Reviewers invited
Invitations sent on 23 Mar, 2021
Submission checks complete
On 13 Mar, 2021
Editor invited
On 13 Mar, 2021
Editor assigned
On 08 Mar, 2021
First submitted
On 08 Mar, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Stem Cell Research & Therapy.
Background
The bone marrow niche supports hematopoietic cell development through intimate contact with multipotent stromal mesenchymal stem cells; however, the intracellular signaling, function, and regulation of such supportive niche cells are still being defined. Our study was designed to understand how G protein receptor kinase 3 (GRK3) affects bone marrow mesenchymal stem cell function by examining primary cells from GRK3-deficient mice, which we have previously published to have a hypercellular bone marrow and leukocytosis through negative regulation of CXCL12/CXCR4 signaling.
Methods
Murine GRK3-deficient bone marrow mesenchymal stromal cells were harvested and cultured to differentiate into three lineages (adipocyte, chondrocyte, and osteoblast) to confirm multi-potency and compared to wild type cells. Immunoblotting, modified-TANGO experiments, and flow cytometry were used to further examine the effects of GRK3 deficiency on bone marrow mesenchymal stromal cell receptor signaling. Microcomputed tomography was used to determine trabecular and cortical bone composition of GRK3-deficient mice and standard ELISA to quantitate CXCL12 production from cellular cultures.
Results
GRK3-deficient, bone marrow-derived mesenchymal stem cells exhibit enhanced and earlier osteogenic differentiation in vitro. The addition of a sphingosine kinase inhibitor abrogated the osteogenic proliferation and differentiation, suggesting sphingosine-1-phosphate receptor signaling was a putative G-protein coupled receptor regulated by GRK3. Immunoblotting showed prolonged ERK1/2 signaling after stimulation with sphingosine-1-phosphate in GRK3-deficient cells, and modified-TANGO assays suggested the involvement of β-arrestin-2 in sphingosine 1 phosphate receptor internalization.
Conclusions
Our work suggests that GRK3 regulates sphingosine-1-phosphate receptor signaling on bone marrow mesenchymal stem cells by recruiting β-arrestin to the occupied GPCR to promote internalization, and lack of such regulation affects mesenchymal stem cell functionality.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
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