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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 differentially expressed gene expression profile for cartilage versus dedifferentiated chondrocytes (dedifferentiation transition). Network consists of genes (nodes) connected by lines (edges) indicating a known relationship/interaction in the IPA® Knowledge Base. Genes more highly expressed in cartilage are coloured red; genes showing low expression in cartilage (i.e. higher in monolayer) are coloured green. Figure prediction legend describes the nature of edges joining nodes. Upstream regulators, and intermediate nodes (Jun, Smad3, Il6, Tnf), were predicted to be activated (orange) or inhibited (blue) consistent with the gene expression profile supplied; the direction and nature of the relationship is also indicated. Based upon the observed gene expression profile Tgf-β1 was predicted to be a key upstream regulator (z-score -2.65, overlap p-value 5.41e-26) of 740 differentially expressed genes; the actions of Tgf-β1 were predicted to be inhibited in native cartilage where expression of Smad7, Bgn, and Ctgf were low relative to monolayer chondrocytes at passage five. Functional annotations for ‘differentiation of chondrocytes’ (3.66e-11), and ‘differentiation of connective tissue cells’ (5.6e-21, inhibited) were significantly enriched for this subnetwork and indicated a differentiation process; (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 evidence of associations are defined in the network legend. Elements determined to influence differentiation status of chondrocytes and tenocytes in culture using IPA® are shown to have significant enrichment for protein-protein interactions (p < 0.0001) indicating that as a group they are biologically connected.
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f5: (a) Hypothetical mechanistic network derived from differentially expressed gene expression profile for cartilage versus dedifferentiated chondrocytes (dedifferentiation transition). Network consists of genes (nodes) connected by lines (edges) indicating a known relationship/interaction in the IPA® Knowledge Base. Genes more highly expressed in cartilage are coloured red; genes showing low expression in cartilage (i.e. higher in monolayer) are coloured green. Figure prediction legend describes the nature of edges joining nodes. Upstream regulators, and intermediate nodes (Jun, Smad3, Il6, Tnf), were predicted to be activated (orange) or inhibited (blue) consistent with the gene expression profile supplied; the direction and nature of the relationship is also indicated. Based upon the observed gene expression profile Tgf-β1 was predicted to be a key upstream regulator (z-score -2.65, overlap p-value 5.41e-26) of 740 differentially expressed genes; the actions of Tgf-β1 were predicted to be inhibited in native cartilage where expression of Smad7, Bgn, and Ctgf were low relative to monolayer chondrocytes at passage five. Functional annotations for ‘differentiation of chondrocytes’ (3.66e-11), and ‘differentiation of connective tissue cells’ (5.6e-21, inhibited) were significantly enriched for this subnetwork and indicated a differentiation process; (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 evidence of associations are defined in the network legend. Elements determined to influence differentiation status of chondrocytes and tenocytes in culture using IPA® are shown to have significant enrichment for protein-protein interactions (p < 0.0001) indicating that as a group they are biologically connected.

Mentions: To develop testable hypothetical mechanistic networks for de- and redifferentiation, upstream master regulators of the observed gene expression profiles for all pairwise comparisons, were inferred using Ingenuity® Pathway Analysis (IPA). The top scoring upstream regulators were ordered by ‘overlap p-value’, a measure of the enrichment of regulated genes within a data set, and by calculated ‘z-score’ for the predicted activation state of a regulator inferred from a test of the match in up- and down-regulation patterns. In all comparisons the top predictions for upstream regulators of the observed synthetic profile were TGF-β, TNF, and MYC. For the dedifferentiation transition in both chondrocytes, Fig. 5a,b, and tenocytes TGF-β mediated effects were predicted to be inhibited in native tissue, i.e. activated in dedifferentiation. Additionally, IL6 was predicted for chondrocytes and HIF1α and PDGF BB for tenocytes.


A systems biology approach to defining regulatory mechanisms for cartilage and tendon cell phenotypes
(a) Hypothetical mechanistic network derived from differentially expressed gene expression profile for cartilage versus dedifferentiated chondrocytes (dedifferentiation transition). Network consists of genes (nodes) connected by lines (edges) indicating a known relationship/interaction in the IPA® Knowledge Base. Genes more highly expressed in cartilage are coloured red; genes showing low expression in cartilage (i.e. higher in monolayer) are coloured green. Figure prediction legend describes the nature of edges joining nodes. Upstream regulators, and intermediate nodes (Jun, Smad3, Il6, Tnf), were predicted to be activated (orange) or inhibited (blue) consistent with the gene expression profile supplied; the direction and nature of the relationship is also indicated. Based upon the observed gene expression profile Tgf-β1 was predicted to be a key upstream regulator (z-score -2.65, overlap p-value 5.41e-26) of 740 differentially expressed genes; the actions of Tgf-β1 were predicted to be inhibited in native cartilage where expression of Smad7, Bgn, and Ctgf were low relative to monolayer chondrocytes at passage five. Functional annotations for ‘differentiation of chondrocytes’ (3.66e-11), and ‘differentiation of connective tissue cells’ (5.6e-21, inhibited) were significantly enriched for this subnetwork and indicated a differentiation process; (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 evidence of associations are defined in the network legend. Elements determined to influence differentiation status of chondrocytes and tenocytes in culture using IPA® are shown to have significant enrichment for protein-protein interactions (p < 0.0001) indicating that as a group they are biologically connected.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037390&req=5

f5: (a) Hypothetical mechanistic network derived from differentially expressed gene expression profile for cartilage versus dedifferentiated chondrocytes (dedifferentiation transition). Network consists of genes (nodes) connected by lines (edges) indicating a known relationship/interaction in the IPA® Knowledge Base. Genes more highly expressed in cartilage are coloured red; genes showing low expression in cartilage (i.e. higher in monolayer) are coloured green. Figure prediction legend describes the nature of edges joining nodes. Upstream regulators, and intermediate nodes (Jun, Smad3, Il6, Tnf), were predicted to be activated (orange) or inhibited (blue) consistent with the gene expression profile supplied; the direction and nature of the relationship is also indicated. Based upon the observed gene expression profile Tgf-β1 was predicted to be a key upstream regulator (z-score -2.65, overlap p-value 5.41e-26) of 740 differentially expressed genes; the actions of Tgf-β1 were predicted to be inhibited in native cartilage where expression of Smad7, Bgn, and Ctgf were low relative to monolayer chondrocytes at passage five. Functional annotations for ‘differentiation of chondrocytes’ (3.66e-11), and ‘differentiation of connective tissue cells’ (5.6e-21, inhibited) were significantly enriched for this subnetwork and indicated a differentiation process; (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 evidence of associations are defined in the network legend. Elements determined to influence differentiation status of chondrocytes and tenocytes in culture using IPA® are shown to have significant enrichment for protein-protein interactions (p < 0.0001) indicating that as a group they are biologically connected.
Mentions: To develop testable hypothetical mechanistic networks for de- and redifferentiation, upstream master regulators of the observed gene expression profiles for all pairwise comparisons, were inferred using Ingenuity® Pathway Analysis (IPA). The top scoring upstream regulators were ordered by ‘overlap p-value’, a measure of the enrichment of regulated genes within a data set, and by calculated ‘z-score’ for the predicted activation state of a regulator inferred from a test of the match in up- and down-regulation patterns. In all comparisons the top predictions for upstream regulators of the observed synthetic profile were TGF-β, TNF, and MYC. For the dedifferentiation transition in both chondrocytes, Fig. 5a,b, and tenocytes TGF-β mediated effects were predicted to be inhibited in native tissue, i.e. activated in dedifferentiation. Additionally, IL6 was predicted for chondrocytes and HIF1α and PDGF BB for tenocytes.

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