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A systems biology approach to defining regulatory mechanisms for cartilage and tendon cell phenotypes

View Article: PubMed Central - PubMed

ABSTRACT

Phenotypic plasticity of adult somatic cells has provided emerging avenues for the development of regenerative therapeutics. In musculoskeletal biology the mechanistic regulatory networks of genes governing the phenotypic plasticity of cartilage and tendon cells has not been considered systematically. Additionally, a lack of strategies to effectively reproduce in vitro functional models of cartilage and tendon is retarding progress in this field. De- and redifferentiation represent phenotypic transitions that may contribute to loss of function in ageing musculoskeletal tissues. Applying a systems biology network analysis approach to global gene expression profiles derived from common in vitro culture systems (monolayer and three-dimensional cultures) this study demonstrates common regulatory mechanisms governing de- and redifferentiation transitions in cartilage and tendon cells. Furthermore, evidence of convergence of gene expression profiles during monolayer expansion of cartilage and tendon cells, and the expression of key developmental markers, challenges the physiological relevance of this culture system. The study also suggests that oxidative stress and PI3K signalling pathways are key modulators of in vitro phenotypes for cells of musculoskeletal origin.

No MeSH data available.


Related in: MedlinePlus

(a) Hypothetical mechanistic network derived from predicted upstream regulators for genes found to be differentially expression in chondrocytes in transition from monolayer to alginate bead cultures (redifferentiation). Genes showing lower expression in monolayer relative to alginate beads (Hif1a, Nfe2l2, Atf3) are coloured green (i.e. indicating higher expression in alginate cultures); genes in orange are predicted. Genes more highly expressed in monolayer are not shown (absence of red-coloured genes). In monolayer chondrocytes PDGF BB mediated effects are predicted to be inactive for the observed expression profile, i.e. activated in alginate cultures. Ingenuity® canonical pathways significantly enriched for this expression profile included ‘NRF2-mediated oxidative stress’, ‘PI3K/AKT signalling’ and ‘the role of osteoblasts, osteoclasts and chondrocytes in rheumatoid arthritis’. The physiological function terms ‘differentiation of cells’ (p = 1.6e-14) and ‘apoptosis’ (p = 4.9e-12) were highly enriched in this subnetwork. Nodes with a pink border were enriched for ‘condensation of cartilage tissue’, p = 2.07e-7. The small molecule inhibitor of the PI3K/AKT pathway ‘LY294004’ was predicted to be activated based upon the supplied gene expression profile (z-score = 4.58, overlap p-value = 3.3e-15, 219 gene interactions). This mechanistic network indicates that PI3K signalling and PDGF BB are likely to be active in the redifferentiation phenotype in alginate cultures; the PI3K inhibitor LY294004 describes the inverse for monolayer cells. (b) Using the same elements a protein-protein association network consisting of nodes (proteins) and edges (evidence of associations) indicates a shared function between selected nodes, but not necessarily physical interactions. Sources for associations are defined in the network legend. Elements determined to influence differentiation status of chondrocytes and tenocytes in culture are shown to have significant enrichment for protein-protein interactions (p < 0.0001) indicating that as a group they are biologically connected. Some elements did not demonstrate evidence of association in this network (GPNMB, CHI3L).
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f6: (a) Hypothetical mechanistic network derived from predicted upstream regulators for genes found to be differentially expression in chondrocytes in transition from monolayer to alginate bead cultures (redifferentiation). Genes showing lower expression in monolayer relative to alginate beads (Hif1a, Nfe2l2, Atf3) are coloured green (i.e. indicating higher expression in alginate cultures); genes in orange are predicted. Genes more highly expressed in monolayer are not shown (absence of red-coloured genes). In monolayer chondrocytes PDGF BB mediated effects are predicted to be inactive for the observed expression profile, i.e. activated in alginate cultures. Ingenuity® canonical pathways significantly enriched for this expression profile included ‘NRF2-mediated oxidative stress’, ‘PI3K/AKT signalling’ and ‘the role of osteoblasts, osteoclasts and chondrocytes in rheumatoid arthritis’. The physiological function terms ‘differentiation of cells’ (p = 1.6e-14) and ‘apoptosis’ (p = 4.9e-12) were highly enriched in this subnetwork. Nodes with a pink border were enriched for ‘condensation of cartilage tissue’, p = 2.07e-7. The small molecule inhibitor of the PI3K/AKT pathway ‘LY294004’ was predicted to be activated based upon the supplied gene expression profile (z-score = 4.58, overlap p-value = 3.3e-15, 219 gene interactions). This mechanistic network indicates that PI3K signalling and PDGF BB are likely to be active in the redifferentiation phenotype in alginate cultures; the PI3K inhibitor LY294004 describes the inverse for monolayer cells. (b) Using the same elements a protein-protein association network consisting of nodes (proteins) and edges (evidence of associations) indicates a shared function between selected nodes, but not necessarily physical interactions. Sources for associations are defined in the network legend. Elements determined to influence differentiation status of chondrocytes and tenocytes in culture are shown to have significant enrichment for protein-protein interactions (p < 0.0001) indicating that as a group they are biologically connected. Some elements did not demonstrate evidence of association in this network (GPNMB, CHI3L).

Mentions: Further evidence of consensus for upstream regulators was evident in analysis of the expression data for the transition between two- and three-dimensional culture systems. For both alginate, Fig. 6a,b, and fibrin cultures the gene expression profiles were consistent with PDGF BB and TNF as activated upstream regulators. Reciprocal activation was predicted for the small molecule inhibitor of PI3K signalling, LY294004, and PI3K activator PDGF BB in 3D cultures, consistent with SPIA prediction of activation of the PI3K pathway in redifferentiation.


A systems biology approach to defining regulatory mechanisms for cartilage and tendon cell phenotypes
(a) Hypothetical mechanistic network derived from predicted upstream regulators for genes found to be differentially expression in chondrocytes in transition from monolayer to alginate bead cultures (redifferentiation). Genes showing lower expression in monolayer relative to alginate beads (Hif1a, Nfe2l2, Atf3) are coloured green (i.e. indicating higher expression in alginate cultures); genes in orange are predicted. Genes more highly expressed in monolayer are not shown (absence of red-coloured genes). In monolayer chondrocytes PDGF BB mediated effects are predicted to be inactive for the observed expression profile, i.e. activated in alginate cultures. Ingenuity® canonical pathways significantly enriched for this expression profile included ‘NRF2-mediated oxidative stress’, ‘PI3K/AKT signalling’ and ‘the role of osteoblasts, osteoclasts and chondrocytes in rheumatoid arthritis’. The physiological function terms ‘differentiation of cells’ (p = 1.6e-14) and ‘apoptosis’ (p = 4.9e-12) were highly enriched in this subnetwork. Nodes with a pink border were enriched for ‘condensation of cartilage tissue’, p = 2.07e-7. The small molecule inhibitor of the PI3K/AKT pathway ‘LY294004’ was predicted to be activated based upon the supplied gene expression profile (z-score = 4.58, overlap p-value = 3.3e-15, 219 gene interactions). This mechanistic network indicates that PI3K signalling and PDGF BB are likely to be active in the redifferentiation phenotype in alginate cultures; the PI3K inhibitor LY294004 describes the inverse for monolayer cells. (b) Using the same elements a protein-protein association network consisting of nodes (proteins) and edges (evidence of associations) indicates a shared function between selected nodes, but not necessarily physical interactions. Sources for associations are defined in the network legend. Elements determined to influence differentiation status of chondrocytes and tenocytes in culture are shown to have significant enrichment for protein-protein interactions (p < 0.0001) indicating that as a group they are biologically connected. Some elements did not demonstrate evidence of association in this network (GPNMB, CHI3L).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037390&req=5

f6: (a) Hypothetical mechanistic network derived from predicted upstream regulators for genes found to be differentially expression in chondrocytes in transition from monolayer to alginate bead cultures (redifferentiation). Genes showing lower expression in monolayer relative to alginate beads (Hif1a, Nfe2l2, Atf3) are coloured green (i.e. indicating higher expression in alginate cultures); genes in orange are predicted. Genes more highly expressed in monolayer are not shown (absence of red-coloured genes). In monolayer chondrocytes PDGF BB mediated effects are predicted to be inactive for the observed expression profile, i.e. activated in alginate cultures. Ingenuity® canonical pathways significantly enriched for this expression profile included ‘NRF2-mediated oxidative stress’, ‘PI3K/AKT signalling’ and ‘the role of osteoblasts, osteoclasts and chondrocytes in rheumatoid arthritis’. The physiological function terms ‘differentiation of cells’ (p = 1.6e-14) and ‘apoptosis’ (p = 4.9e-12) were highly enriched in this subnetwork. Nodes with a pink border were enriched for ‘condensation of cartilage tissue’, p = 2.07e-7. The small molecule inhibitor of the PI3K/AKT pathway ‘LY294004’ was predicted to be activated based upon the supplied gene expression profile (z-score = 4.58, overlap p-value = 3.3e-15, 219 gene interactions). This mechanistic network indicates that PI3K signalling and PDGF BB are likely to be active in the redifferentiation phenotype in alginate cultures; the PI3K inhibitor LY294004 describes the inverse for monolayer cells. (b) Using the same elements a protein-protein association network consisting of nodes (proteins) and edges (evidence of associations) indicates a shared function between selected nodes, but not necessarily physical interactions. Sources for associations are defined in the network legend. Elements determined to influence differentiation status of chondrocytes and tenocytes in culture are shown to have significant enrichment for protein-protein interactions (p < 0.0001) indicating that as a group they are biologically connected. Some elements did not demonstrate evidence of association in this network (GPNMB, CHI3L).
Mentions: Further evidence of consensus for upstream regulators was evident in analysis of the expression data for the transition between two- and three-dimensional culture systems. For both alginate, Fig. 6a,b, and fibrin cultures the gene expression profiles were consistent with PDGF BB and TNF as activated upstream regulators. Reciprocal activation was predicted for the small molecule inhibitor of PI3K signalling, LY294004, and PI3K activator PDGF BB in 3D cultures, consistent with SPIA prediction of activation of the PI3K pathway in redifferentiation.

View Article: PubMed Central - PubMed

ABSTRACT

Phenotypic plasticity of adult somatic cells has provided emerging avenues for the development of regenerative therapeutics. In musculoskeletal biology the mechanistic regulatory networks of genes governing the phenotypic plasticity of cartilage and tendon cells has not been considered systematically. Additionally, a lack of strategies to effectively reproduce in vitro functional models of cartilage and tendon is retarding progress in this field. De- and redifferentiation represent phenotypic transitions that may contribute to loss of function in ageing musculoskeletal tissues. Applying a systems biology network analysis approach to global gene expression profiles derived from common in vitro culture systems (monolayer and three-dimensional cultures) this study demonstrates common regulatory mechanisms governing de- and redifferentiation transitions in cartilage and tendon cells. Furthermore, evidence of convergence of gene expression profiles during monolayer expansion of cartilage and tendon cells, and the expression of key developmental markers, challenges the physiological relevance of this culture system. The study also suggests that oxidative stress and PI3K signalling pathways are key modulators of in vitro phenotypes for cells of musculoskeletal origin.

No MeSH data available.


Related in: MedlinePlus