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Sensitivity analysis of intracellular signaling pathway kinetics predicts targets for stem cell fate control.

Mahdavi A, Davey RE, Bhola P, Yin T, Zandstra PW - PLoS Comput. Biol. (2007)

Bottom Line: Our analysis identified novel pathway responses; for example, overexpression of the receptor glycoprotein-130 results in reduced pathway activation and increased ESC differentiation.Our analysis demonstrates that signaling activation and desensitization (the inability to respond to ligand restimulation) is regulated by balancing the activation state of a distributed set of parameters including nuclear export of Stat3, nuclear phosphatase activity, inhibition by suppressor of cytokine signaling, and receptor trafficking.This knowledge was used to devise a temporally modulated ligand delivery strategy that maximizes signaling activation and leads to enhanced ESC self-renewal.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Directing stem cell fate requires knowledge of how signaling networks integrate temporally and spatially segregated stimuli. We developed and validated a computational model of signal transducer and activator of transcription-3 (Stat3) pathway kinetics, a signaling network involved in embryonic stem cell (ESC) self-renewal. Our analysis identified novel pathway responses; for example, overexpression of the receptor glycoprotein-130 results in reduced pathway activation and increased ESC differentiation. We used a systematic in silico screen to identify novel targets and protein interactions involved in Stat3 activation. Our analysis demonstrates that signaling activation and desensitization (the inability to respond to ligand restimulation) is regulated by balancing the activation state of a distributed set of parameters including nuclear export of Stat3, nuclear phosphatase activity, inhibition by suppressor of cytokine signaling, and receptor trafficking. This knowledge was used to devise a temporally modulated ligand delivery strategy that maximizes signaling activation and leads to enhanced ESC self-renewal.

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Desensitization of ESCs to LIF Stimulation Can Be Investigated Computationally and Used to Control Self-Renewal(A) Model-predicted trends of desensitization of Stat3 activation to LIF stimulation. Color bar inset shows history of 500 pM LIF stimulations.(B) Experimental results are in agreement with model-predicted desensitization kinetics in (A) and show a gradual loss of desensitization in absence of ligand.(C) Global sensitivity analysis shows parameter interactions which result in right-shift (positive minutes) or left-shift (negative minutes) in restimulation profile of Stat3. Shift in the profile was determined from the maximum of cross-correlation of two consecutive Stat3 activation profiles.(D) Sensitivity analysis clustergram shows Euclidean distance measurement between two consecutive Stat3 activation profiles and distinguishes parameter interactions which influence desensitization by changing the restimulation profile.(E) Experimental results of dose response of Stat3 activation to LIF show the steady state response as well as the peak values of Stat3 activation during transient LIF stimulation.(F) Dose response of Oct4 expression after 72 h at different LIF concentrations.(G) Stat3 activation profiles for 10 pM LIF stimulation with 1-h and 6-h periods in red and blue, respectively. During 6-h cycles, LIF is removed for 3 h during which time a lower Stat3 activation level is observed in comparison with the 1-h period.(H) Histograms of Oct4 expression show that 1-hr period of LIF stimulation (red) maintains a higher percentage of Oct4+ cells than 6-h period of LIF stimulation after 72 h.(I) Oct4 expression of cells maintained for 72 h in different conditions shows that the percentage of Oct4+ cells in the 1-h period is higher than the 6-h period and comparable to 500 pM constant LIF levels.
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pcbi-0030130-g006: Desensitization of ESCs to LIF Stimulation Can Be Investigated Computationally and Used to Control Self-Renewal(A) Model-predicted trends of desensitization of Stat3 activation to LIF stimulation. Color bar inset shows history of 500 pM LIF stimulations.(B) Experimental results are in agreement with model-predicted desensitization kinetics in (A) and show a gradual loss of desensitization in absence of ligand.(C) Global sensitivity analysis shows parameter interactions which result in right-shift (positive minutes) or left-shift (negative minutes) in restimulation profile of Stat3. Shift in the profile was determined from the maximum of cross-correlation of two consecutive Stat3 activation profiles.(D) Sensitivity analysis clustergram shows Euclidean distance measurement between two consecutive Stat3 activation profiles and distinguishes parameter interactions which influence desensitization by changing the restimulation profile.(E) Experimental results of dose response of Stat3 activation to LIF show the steady state response as well as the peak values of Stat3 activation during transient LIF stimulation.(F) Dose response of Oct4 expression after 72 h at different LIF concentrations.(G) Stat3 activation profiles for 10 pM LIF stimulation with 1-h and 6-h periods in red and blue, respectively. During 6-h cycles, LIF is removed for 3 h during which time a lower Stat3 activation level is observed in comparison with the 1-h period.(H) Histograms of Oct4 expression show that 1-hr period of LIF stimulation (red) maintains a higher percentage of Oct4+ cells than 6-h period of LIF stimulation after 72 h.(I) Oct4 expression of cells maintained for 72 h in different conditions shows that the percentage of Oct4+ cells in the 1-h period is higher than the 6-h period and comparable to 500 pM constant LIF levels.

Mentions: Desensitization, which is the inability to respond to ligand restimulation [26], may significantly limit cytokine-driven in vitro stem cell propagation [43]. Possible mechanisms of desensitization in the Jak/Stat3 pathway include receptor downregulation, SHP2- or SOCS3-mediated signal attenuation, and differential transport kinetics of STAT3, but the relative importance of each interaction is unknown. As a first step to investigate this response, model-predicted trends of desensitization (Figure 6A) were experimentally verified (Figure 6B), and showed that a minimum time of about 3 h is required after ligand stimulation for the cells to become fully responsive to the readdition of LIF. GSA of two successive Stat3 activation profiles was used to understand what pathway components control desensitization. This method distinguishes between desensitization and inhibition, since both successive activation profiles will be changed by inhibition, but only the reactivation profile is influenced by desensitization. The relative shift of the reactivation profile of Stat3 (Figure 6C), and the sensitivity analysis (Figure 6D), both showed that the decreased production of SOCS3 and a reduction of Stat3 nuclear export (together or independently) could delay the Stat3 reactivation profile. The main determinants of desensitization control, in order of importance, were SOCS3, nuclear phosphatase, and nuclear export of Stat3. Our analysis also suggested that receptor turnover rates influence desensitization.


Sensitivity analysis of intracellular signaling pathway kinetics predicts targets for stem cell fate control.

Mahdavi A, Davey RE, Bhola P, Yin T, Zandstra PW - PLoS Comput. Biol. (2007)

Desensitization of ESCs to LIF Stimulation Can Be Investigated Computationally and Used to Control Self-Renewal(A) Model-predicted trends of desensitization of Stat3 activation to LIF stimulation. Color bar inset shows history of 500 pM LIF stimulations.(B) Experimental results are in agreement with model-predicted desensitization kinetics in (A) and show a gradual loss of desensitization in absence of ligand.(C) Global sensitivity analysis shows parameter interactions which result in right-shift (positive minutes) or left-shift (negative minutes) in restimulation profile of Stat3. Shift in the profile was determined from the maximum of cross-correlation of two consecutive Stat3 activation profiles.(D) Sensitivity analysis clustergram shows Euclidean distance measurement between two consecutive Stat3 activation profiles and distinguishes parameter interactions which influence desensitization by changing the restimulation profile.(E) Experimental results of dose response of Stat3 activation to LIF show the steady state response as well as the peak values of Stat3 activation during transient LIF stimulation.(F) Dose response of Oct4 expression after 72 h at different LIF concentrations.(G) Stat3 activation profiles for 10 pM LIF stimulation with 1-h and 6-h periods in red and blue, respectively. During 6-h cycles, LIF is removed for 3 h during which time a lower Stat3 activation level is observed in comparison with the 1-h period.(H) Histograms of Oct4 expression show that 1-hr period of LIF stimulation (red) maintains a higher percentage of Oct4+ cells than 6-h period of LIF stimulation after 72 h.(I) Oct4 expression of cells maintained for 72 h in different conditions shows that the percentage of Oct4+ cells in the 1-h period is higher than the 6-h period and comparable to 500 pM constant LIF levels.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1913098&req=5

pcbi-0030130-g006: Desensitization of ESCs to LIF Stimulation Can Be Investigated Computationally and Used to Control Self-Renewal(A) Model-predicted trends of desensitization of Stat3 activation to LIF stimulation. Color bar inset shows history of 500 pM LIF stimulations.(B) Experimental results are in agreement with model-predicted desensitization kinetics in (A) and show a gradual loss of desensitization in absence of ligand.(C) Global sensitivity analysis shows parameter interactions which result in right-shift (positive minutes) or left-shift (negative minutes) in restimulation profile of Stat3. Shift in the profile was determined from the maximum of cross-correlation of two consecutive Stat3 activation profiles.(D) Sensitivity analysis clustergram shows Euclidean distance measurement between two consecutive Stat3 activation profiles and distinguishes parameter interactions which influence desensitization by changing the restimulation profile.(E) Experimental results of dose response of Stat3 activation to LIF show the steady state response as well as the peak values of Stat3 activation during transient LIF stimulation.(F) Dose response of Oct4 expression after 72 h at different LIF concentrations.(G) Stat3 activation profiles for 10 pM LIF stimulation with 1-h and 6-h periods in red and blue, respectively. During 6-h cycles, LIF is removed for 3 h during which time a lower Stat3 activation level is observed in comparison with the 1-h period.(H) Histograms of Oct4 expression show that 1-hr period of LIF stimulation (red) maintains a higher percentage of Oct4+ cells than 6-h period of LIF stimulation after 72 h.(I) Oct4 expression of cells maintained for 72 h in different conditions shows that the percentage of Oct4+ cells in the 1-h period is higher than the 6-h period and comparable to 500 pM constant LIF levels.
Mentions: Desensitization, which is the inability to respond to ligand restimulation [26], may significantly limit cytokine-driven in vitro stem cell propagation [43]. Possible mechanisms of desensitization in the Jak/Stat3 pathway include receptor downregulation, SHP2- or SOCS3-mediated signal attenuation, and differential transport kinetics of STAT3, but the relative importance of each interaction is unknown. As a first step to investigate this response, model-predicted trends of desensitization (Figure 6A) were experimentally verified (Figure 6B), and showed that a minimum time of about 3 h is required after ligand stimulation for the cells to become fully responsive to the readdition of LIF. GSA of two successive Stat3 activation profiles was used to understand what pathway components control desensitization. This method distinguishes between desensitization and inhibition, since both successive activation profiles will be changed by inhibition, but only the reactivation profile is influenced by desensitization. The relative shift of the reactivation profile of Stat3 (Figure 6C), and the sensitivity analysis (Figure 6D), both showed that the decreased production of SOCS3 and a reduction of Stat3 nuclear export (together or independently) could delay the Stat3 reactivation profile. The main determinants of desensitization control, in order of importance, were SOCS3, nuclear phosphatase, and nuclear export of Stat3. Our analysis also suggested that receptor turnover rates influence desensitization.

Bottom Line: Our analysis identified novel pathway responses; for example, overexpression of the receptor glycoprotein-130 results in reduced pathway activation and increased ESC differentiation.Our analysis demonstrates that signaling activation and desensitization (the inability to respond to ligand restimulation) is regulated by balancing the activation state of a distributed set of parameters including nuclear export of Stat3, nuclear phosphatase activity, inhibition by suppressor of cytokine signaling, and receptor trafficking.This knowledge was used to devise a temporally modulated ligand delivery strategy that maximizes signaling activation and leads to enhanced ESC self-renewal.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Directing stem cell fate requires knowledge of how signaling networks integrate temporally and spatially segregated stimuli. We developed and validated a computational model of signal transducer and activator of transcription-3 (Stat3) pathway kinetics, a signaling network involved in embryonic stem cell (ESC) self-renewal. Our analysis identified novel pathway responses; for example, overexpression of the receptor glycoprotein-130 results in reduced pathway activation and increased ESC differentiation. We used a systematic in silico screen to identify novel targets and protein interactions involved in Stat3 activation. Our analysis demonstrates that signaling activation and desensitization (the inability to respond to ligand restimulation) is regulated by balancing the activation state of a distributed set of parameters including nuclear export of Stat3, nuclear phosphatase activity, inhibition by suppressor of cytokine signaling, and receptor trafficking. This knowledge was used to devise a temporally modulated ligand delivery strategy that maximizes signaling activation and leads to enhanced ESC self-renewal.

Show MeSH
Related in: MedlinePlus