Functional Insights in PLS3-Mediated Osteogenic Regulation
Abstract
:1. Introduction
2. Materials and Methods
2.1. In Vivo Characterization of pls3 Knockdown Zebrafish Compensated with Actin-Bundling Proteins
2.2. Acquisition of Human Material
2.3. Cell Culture and Osteogenic Transdifferentiation
2.4. RNA Isolation, cDNA Synthesis, and qPCR
2.5. Western Blotting Analysis
2.6. Pls3 Knockdown in Murine MLO-Y4 Cell Line
2.7. RNA Extraction, cDNA Library Establishment, and Illumina Sequencing
2.8. RNA Sequence Data Analysis
2.9. Mineralization Assay
2.10. Statistical Analysis
3. Results
3.1. Actinin-1 and Actinin-4 Rescue Malformations of Craniofacial Bone Structure, Body Axis, and Tail in 5-Day-Old col1a1-eGFP Zebrafish with pls3-Mediated Morpholino Knockdown
3.2. PLS3 Expression in Fibroblasts of PLS3-Variant Osteoporosis Patients
3.3. Actin-Bundling Protein Expression in PLS3-Variant Patient Fibroblasts and Osteoblast-like Cells
3.4. Pls3 Knockdown in MLO-Y4 Cells
3.5. Differential Gene Expression in MLO-Y4 Cells Following Pls3 Knockdown
3.6. Osteogenic Transdifferentiation Potential of PLS3-Variant Patient Fibroblasts
3.7. Gene Expression of Proteins Involved in WNTSignaling
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Codes | Gender | Age at Biopsy | Mutation | Mutation Type | Low-Impact Peripheral Fractures | Multiple Vertebral Fractures | Other Clinical Findings |
---|---|---|---|---|---|---|---|
C1 | male | 4 | NA | NA | - | - | - |
C2 | male | 45 | NA | NA | - | - | - |
C3 | female | 54 | NA | NA | - | - | - |
C4 | female | 45 | NA | NA | - | - | - |
C5 | male | 47 | NA | NA | - | - | - |
P1 | male | 10 | PLS3: c.235del p.(Tyr79fs) | Frameshift | 6 | No | Acute lymphatic leukemia |
P2 | male | 32 | PLS3: c.235del p.(Tyr79fs) | Frameshift | 13 | Yes | None |
P3 | male | 10 | PLS3: c.235del p.(Tyr79fs) | Frameshift | Multiple | No | Epilepsy and, in childhood, waddling gait |
P4 | male | 6 | PLS3: c.235del p.(Tyr79fs) | Frameshift | 17 | No | Patent ductus arteriosus and, in childhood, waddling gait |
P5 | male | 47 | PLS3: c.748+1G>A | Splice site variant | Multiple | Yes | Alcohol abuse and esophageal carcinoma |
P6 | male | 54 | PLS3: c.759_760insAAT | Insertion | 1 | Yes | None |
Gene | Accession Number | Forward Primer (5′–3′) | Reverse Primer (5′–3′) |
---|---|---|---|
PLS3 | NM_005032.4 | CTCCCTGGTTGGCATTGGAG | GCCAAACTGGAGCTGATCGT |
ACTN1 | NM_001130004.1 | CCCGAGCTGATTGACTACGG | ATGGCTTTCTCATCCGGTCG |
ACTN4 | NM_004924.5 | GGCACAGACCAGAGCTGATT | AAGCGTGGTAGAAGCAGGAC |
FSCN1 | NM_003088.3 | GTCGACTCGCTCATCACCCTC | GGAAGGTCTCCTGGTCGGTC |
RUNX2 | NM_001024630.3 | ACTGCTTGCAGCCTTAAAT | ATGCTTCATTCGCCTCAC |
ALP | NM_000478.5 | AGGGACATTGACGTGATCAT | CCTGGCTCGAAGAGACC |
OPN | NM_001040058.1 | TTCCAAGTAAGTCCAACGAAAG | GTGACCAGTTCATCAGATTCAT |
SFRP1 | NM_003013.2 | CGTGGGCTACAAGAAGATGG | AAGCCGAAGAACTGCATGAC |
WNT1 | NM_005430.3 | GCCGATGGTGGGGTATTGTG | GATCCCCGGATTTTGGCGTA |
CTNND2 | NM_001332.3 | CCGGGTGCCTAAGGAATGTT | CGCAGACTGGATCACGTACA |
WNT2 | NM_003391.2 | CATCCAGATGTGATGCGTGC | GCAGATTCCCGACTACTTCG |
NFATC2 | NM_012340.4 | CATGAGGGCAACCATCGACT | CCCCGTGAGGATCATTTGCT |
NFATC1 | NM_006162.4 | AACCCCATCGAATGCTCCCAG | AGAAACTGACGTGAACGGGG |
FZD3 | NM_017412.3 | CGCCGGGGTCTGAGATATT | GCTTGGTCTCACAAAAGGCG |
GAPDH | NM_002046.6 | GCCCAATACGACCAAATCC | AGCCACATCGCTCAGACAC |
Pls3 | NM_145629.1 | CAAGCACAATAACGCCAAGTACGC | ACTCTCTTCATCCCTCTGCCCATC |
Tbp | NM_013684.2 | TGGCGGTTTGGCTAGGTTTC | CACCATGAAATAGTGATGCTGGG |
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Zhong, W.; Neugebauer, J.; Pathak, J.L.; Li, X.; Pals, G.; Zillikens, M.C.; Eekhoff, E.M.W.; Bravenboer, N.; Zhang, Q.; Hammerschmidt, M.; et al. Functional Insights in PLS3-Mediated Osteogenic Regulation. Cells 2024, 13, 1507. https://doi.org/10.3390/cells13171507
Zhong W, Neugebauer J, Pathak JL, Li X, Pals G, Zillikens MC, Eekhoff EMW, Bravenboer N, Zhang Q, Hammerschmidt M, et al. Functional Insights in PLS3-Mediated Osteogenic Regulation. Cells. 2024; 13(17):1507. https://doi.org/10.3390/cells13171507
Chicago/Turabian StyleZhong, Wenchao, Janine Neugebauer, Janak L. Pathak, Xingyang Li, Gerard Pals, M. Carola Zillikens, Elisabeth M. W. Eekhoff, Nathalie Bravenboer, Qingbin Zhang, Matthias Hammerschmidt, and et al. 2024. "Functional Insights in PLS3-Mediated Osteogenic Regulation" Cells 13, no. 17: 1507. https://doi.org/10.3390/cells13171507