Identification of Impacted Pathways and Transcriptomic Markers as Potential Mediators of Pulmonary Fibrosis in Transgenic Mice Expressing Human IGFBP5
Abstract
:1. Introduction
2. Results
2.1. Transcriptomic Signature and Functional Enrichment of hIGFBP5 pFBs
2.2. Hub Gene Analysis
2.3. Sex Affects the Impact of hIGFBP5 on the mRNA Levels of Genes of Interest in pFBs
2.4. Effect of hIGFBP5 on Intracellular Protein Abundance
2.5. Effect of hIGFBP5 on the Secretion of Igfbp4, Serpine1 and Pappa2
3. Discussion
3.1. Effect of hIGFBP5 on ECM Macromolecules and ECM-Receptor Interaction
3.2. Effect of hIGFBP5 on AGE-RAGE and Egr1-MAPK Dependent Signaling Pathways
3.3. Effect of hIGFBP5 on Focal Adhesion, Calcium Signaling and Actin Polymerization
3.4. Effect of hIGFBP5 on PI3K-Akt, Cell Survival, and Angiogenesis
3.5. hIGFBP5 Downregulated Igfbp3, Igfbp4 and Igfbp7
3.6. Effect of hIGFBP5 on Neuroactive Ligand-Receptor Interaction
3.7. Overall Impact on Receptors and Integrins
3.8. Downregulation of Bdkrb2, one of the Most Relevant Hub Genes
3.9. Effect of hIGFBP5 on Chemokine and Cytokine Signaling
3.10. Endogenous vs. Exogenous IGFBP5
3.11. Sex Differences in the Response of Fibroblasts
3.12. Conclusions
4. Materials and Methods
4.1. Ethics Statement
4.2. Culture of Primary Mouse Lung Fibroblasts
4.3. RNA Extraction and Preparation
4.4. RNA Sequencing & Differential Expression Analysis
4.5. Functional Enrichment & Hub Gene Network Analysis
4.6. Quantitative Reverse Transcription (qRT-PCR)
4.7. Immunoblotting
4.8. Ponceau S Staining
4.9. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Conflicts of Interest
References
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Category | Name | Count DEG | Count All | p-Value | Correction |
---|---|---|---|---|---|
BP | Angiogenesis | 134 | 446 | 3.67E-04 | SCDP |
BP | positive regulation of MAPK cascade | 117 | 412 | 8.15E-03 | SCDP |
BP | regulation of blood coagulation | 28 | 62 | 1.32E-02 | SCDP |
BP | regulation of cell migration | 217 | 798 | 2.55E-02 | SCDP |
BP | morphogenesis of a branching structure | 70 | 199 | 3.05E-02 | SCDP |
BP | positive regulation of cell population proliferation | 190 | 764 | 4.07E-02 | SCDP |
BP | biological adhesion | 287 | 1046 | 4.30E-02 | SCDP |
BP | Ossification | 88 | 312 | 4.30E-02 | SCDP |
BP | response to gonadotropin | 8 | 10 | 4.30E-02 | SCDP |
MF | calcium ion binding | 111 | 411 | 1.71E-02 | highSP |
MF | cytokine activity | 39 | 110 | 1.71E-02 | highSP |
MF | growth factor activity | 33 | 91 | 2.96E-02 | highSP |
MF | receptor ligand activity | 81 | 246 | 5.69E-05 | SCDP |
MF | calcium ion binding | 111 | 411 | 8.65E-03 | SCDP |
MF | carbohydrate binding | 58 | 185 | 9.67E-03 | SCDP |
MF | cell adhesion molecule binding | 60 | 195 | 9.67E-03 | SCDP |
MF | extracellular matrix structural constituent | 39 | 117 | 3.03E-02 | SCDP |
MF | glycosaminoglycan binding | 48 | 154 | 3.03E-02 | SCDP |
CC | extracellular space | 271 | 867 | 9.14E-17 | highSP |
CC | integral component of plasma membrane | 231 | 819 | 4.75E-08 | highSP |
CC | cell surface | 189 | 625 | 9.10E-08 | highSP |
CC | extracellular matrix | 122 | 377 | 1.04E-05 | highSP |
CC | integral component of membrane | 749 | 3218 | 3.56E-05 | highSP |
CC | collagen-containing extracellular matrix | 85 | 293 | 2.37E-03 | highSP |
CC | external side of plasma membrane | 75 | 266 | 2.37E-02 | highSP |
CC | Z disc | 32 | 95 | 3.71E-02 | highSP |
CC | extracellular region | 395 | 1362 | 7.12E-18 | SCDP |
CC | intrinsic component of membrane | 770 | 3318 | 1.96E-12 | SCDP |
CC | cell surface | 189 | 625 | 4.75E-11 | SCDP |
CC | apical part of cell | 91 | 318 | 1.25E-03 | SCDP |
CC | I band | 35 | 103 | 2.61E-02 | SCDP |
PW | Cytokine–cytokine receptor interaction | 52 | 163 | 6.45E-07 | none |
PW | Focal adhesion | 47 | 187 | 2.46E-05 | none |
PW | Staphylococcus aureus infection | 14 | 37 | 3.04E-05 | none |
PW | Viral protein interaction with cytokine and cytokine receptor | 18 | 50 | 6.95E-05 | none |
PW | AGE-RAGE signaling pathway in diabetic complications | 34 | 98 | 1.29E-04 | none |
PW | Systemic lupus erythematosus | 20 | 77 | 1.97E-04 | none |
PW | Proteoglycans in cancer | 54 | 186 | 2.79E-04 | none |
PW | Chemokine signaling pathway | 35 | 147 | 7.48E-04 | none |
PW | Neuroactive ligand-receptor interaction | 38 | 133 | 1.10E-03 | none |
PW | Calcium signaling pathway | 34 | 150 | 2.06E-03 | none |
Pathways | Genes | p-Value | Outcomes |
---|---|---|---|
Cytokine–cytokine receptor interaction | Cxcr4, Ccl20, Ccl28 | 6.45E-07 | Innate and adaptive inflammatory host defenses, cell growth, differentiation, cell death, angiogenesis, development and repair processes aimed at the restoration of homeostasis |
Chemokine signaling pathway | Cxcr4, Ccl20, Ccl28 | 7.48E-04 | Inflammatory immune response, cellular activation, differentiation and survival, cellular polarization and actin reorganization |
Neuroactive ligand-receptor interaction | Fpr1, Bdkrb2, Mchr1, Nmur1, Cnr2, P2ry14, Ptger3 | 1.10E-03 | Environmental information processing |
Calcium signaling pathway | Gna15, Bdkrb2, Cxcr4, Ptger3 | 2.06E-03 | MAPK signaling, apoptosis, long-term potentiation/depression, phosphatidylinositol signaling pathway, contraction, metabolism, proliferation |
Axon guidance | Cxcr4 | 2.15E-03 | Formation of neuronal network, cytoskeletal organization |
Complement and coagulation cascades | Bdkrb2 | 4.48E-03 | Innate immunity, recruitment of inflammatory and immunocompetent cells |
cGMP-PKG signaling pathway | Bdkrb2 | 1.98E-02 | Physiologic processes, regulation of cytosolic calcium concentration and sensitivity of myofilaments to Ca2+, ROS release from mitochondria |
Regulation of actin cytoskeleton | Bdkrb2 | 2.92E-02 | Focal adhesion, MAPK signaling, adherens junction, |
Endocrine and other factor-regulated calcium reabsorption | Bdkrb2 | 4.33E-02 | Intracellular signalling processes, neuronal excitability, muscle contraction and bone formation |
Rap1 signaling pathway | Fpr1 | 5.01E-03 | Cell adhesion, cell–cell junction formation and cell polarity, control of cellcell and cell-matrix interactions by regulating the function of integrins and other adhesion molecules, MAPK signaling |
TNF signaling pathway | Ccl20 | 2.71E-02 | Apoptosis and cell survival as well as inflammation and immunity, MAPK cascade, apoptosis, necroptosis, PI3K-dependent NF-kappa B pathway, JNK pathway, survival. |
cAMP signaling pathway | Hcar2, Ptger3 | 0.487 | Metabolism, secretion, calcium homeostasis, muscle contraction, cell fate, and gene transcription |
Genes | Full Name | RNAseq (F) | qRT-PCR | Immunoblot Cell Lysates | Immunoblot Cond. Media | |
---|---|---|---|---|---|---|
Log2Fc | q-Value | Regulation | Regulation | Regulation | ||
Col27a1 | collagen, type XXVII, alpha 1 | 3.201 | 1.00E-06 | Up | NA | NA |
Pcolce2 | procollagen C-endopeptidase enhancer 2 | 3.124 | 1.12E-03 | Up | NA | NA |
Pappa2 | pappalysin 2 | 1.351 | 2.00E-03 | NA | NA | Up |
Nmur1 | neuromedin U receptor 1 | −3.449 | 4.61E-02 | Down | Down | NA |
Il6 | interleukin 6 | −3.021 | 1.00E-06 | Down | NA | NA |
Bdkrb2 | bradykinin receptor, beta 2 | −5.230 | 5.77E-04 | Down | Down | NA |
Cav3 | caveolin 3 | −7.164 | 1.00E-06 | Down | NA | NA |
Spp1 | secreted phosphoprotein 1 | −3.034 | 2.21E-02 | Down | Down | NA |
Serpine1 | serpin family E member 1 | −2.515 | 1.00E-06 | NA | Down | Down |
Fpr1 | formyl peptide receptor 1 | −5.713 | 7.96E-03 | NA | Down | NA |
Tbx1 | T-box 1 | −9.079 | 4.20E-02 | NA | Down | NA |
Tgfβ2 | transforming growth factor, beta 2 | −1.178 | 1.00E-06 | NA | Down | NA |
Igfbp4 | insulin-like growth factor binding protein 4 | −3.539 | 1.00E-06 | NA | NA | Down |
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Nguyen, X.-X.; Renaud, L.; Feghali-Bostwick, C. Identification of Impacted Pathways and Transcriptomic Markers as Potential Mediators of Pulmonary Fibrosis in Transgenic Mice Expressing Human IGFBP5. Int. J. Mol. Sci. 2021, 22, 12609. https://doi.org/10.3390/ijms222212609
Nguyen X-X, Renaud L, Feghali-Bostwick C. Identification of Impacted Pathways and Transcriptomic Markers as Potential Mediators of Pulmonary Fibrosis in Transgenic Mice Expressing Human IGFBP5. International Journal of Molecular Sciences. 2021; 22(22):12609. https://doi.org/10.3390/ijms222212609
Chicago/Turabian StyleNguyen, Xinh-Xinh, Ludivine Renaud, and Carol Feghali-Bostwick. 2021. "Identification of Impacted Pathways and Transcriptomic Markers as Potential Mediators of Pulmonary Fibrosis in Transgenic Mice Expressing Human IGFBP5" International Journal of Molecular Sciences 22, no. 22: 12609. https://doi.org/10.3390/ijms222212609