Research ArticleSystems Biology

Network-level effects of kinase inhibitors modulate TNF-α–induced apoptosis in the intestinal epithelium

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Science Signaling  15 Dec 2015:
Vol. 8, Issue 407, pp. ra129
DOI: 10.1126/scisignal.aac7235
  • Fig. 1 MAPK pathway inhibitors yield distinct phenotypic and signaling profiles in the mouse intestinal epithelium.

    (A to C) Mice were pretreated (as described in Materials and Methods) with dimethyl sulfoxide (DMSO; as a vehicle control), the MEK inhibitor PD325901 (PD), the ERK inhibitor SCH772984 (SCH), or the Raf inhibitor MLN2480 (MLN), as indicated, before being treated with TNF-α for the indicated times. Intestinal epithelia from the mice were then analyzed by quantitative Western blotting or Bio-Plex to determine the relative abundance of (A) CC3, normalized to that of tubulin, as an indicator of the extent of apoptosis (see Table 1); (B) pERK, normalized to that of total ERK; and (C) pMEK, normalized to that of total MEK. Data in (A) are means ± SEM of two to four experiments per time point, whereas data in (B) and (C) are means ± SEM of three to five experiments per time point. a.u., arbitrary unit.

  • Fig. 2 Mechanistically distinct MEK inhibitors have differential effects on apoptosis and signaling responses.

    (A to C) Mice were pretreated with DMSO, the MEK inhibitor PD325901 (PD), or the MEK inhibitor CH5126766 (CH), as indicated, before being treated with TNF-α for the indicated times. Intestinal epithelia from the mice were then analyzed by quantitative Western blotting or Bio-Plex to determine the relative abundance of (A) CC3, normalized to that of tubulin; (B) pERK, normalized to that of total ERK; and (C) pMEK, normalized to that of total MEK. Data are means ± SEM of two to four experiments per time point.

  • Fig. 3 Measurement of multiple signaling nodes governing cell fate demonstrates signaling differences that are intrinsic and extrinsic to the MAPK pathway in response to PD325901 and CH5126766.

    (A) Illustration of the signaling network governing cell fate and the phosphoprotein nodes measured by Bio-Plex (highlighted in orange). (B) Mice were pretreated with DMSO, the MEK inhibitor PD325901 (PD), or the MEK inhibitor CH5126766 (CH), as indicated, before being treated with TNF-α for the indicated times. Intestinal epithelia from the mice were then analyzed by Bio-Plex to determine the relative abundances of the indicated phosphoproteins. Data in all panels are means ± SEM of two to four experiments per time point.

  • Fig. 4 Computational models identify signaling outside of the MAPK pathway that distinguishes the apoptotic response to MEK inhibitors.

    (A) Left: D-PLSR analysis by grouping all time points and replicates from each condition revealed an axis (LV1) that distinguishes the CH5126766-treated condition from the DMSO- and PD325901-treated conditions. Right: LV1 is composed of signals (phosphoproteins) that were increased or decreased in abundance in the CH5126766-treated conditions relative to those in the DMSO- and PD325901-treated conditions. (B) An SOM was used to identify signaling pathways that clustered across temporal and conditional space. The SOM produced a nodal distance matrix that yielded at least three clear valleys: upper left, upper right, and lower left. (C) A k-means clustering of the distance matrix. Each signaling group corresponds to a distinct temporal activation profile for each condition. Group 3, which corresponds to Akt and Atf-2 signaling, is highly up-regulated in CH5126766 relative to both the DMSO and PD325901 conditions. Signals projected into different groups from replicates (1) and (2) (fig. S3B) are indicated.

  • Fig. 5 Global network shifts govern the apoptotic response to the combinatorial application of MEK and Akt inhibitors.

    (A and B) Mice were pretreated with the MEK inhibitor PD325901 (PD) or the MEK inhibitor CH5126766 (CH) in the presence or absence of the PI3K inhibitor NVP-BEZ235 (BEZ), as indicated, before being treated with TNF-α for the indicated times. Intestinal epithelia from the mice were then analyzed by quantitative Western blotting or Bio-Plex to determine (A) the relative abundance of CC3, normalized to that of tubulin, as an indicator of apoptosis, and (B) the relative abundances of the indicated phosphoproteins. Data in (A) are means ± SEM of three experiments per time point, whereas data in (B) are means ± SEM of three or four experiments per time point. (C) The computational D-PLSR model derived from the DMSO-, CH5126766-, and PD325901-treated conditions (from Fig. 3B). X’s and ellipses represent centroids and 95% confidence intervals (CIs) (34) for the CH and CH + BEZ treatment groups, respectively. (D) Mice were pretreated with the MEK inhibitor CH5126766 (CH) in the presence or absence of the PI3K inhibitor NVP-BEZ235 (BEZ) or the Akt inhibitor MK-2206 (MK), as indicated, before being treated with TNF-α for the indicated times. Intestinal epithelia from the mice were then analyzed by quantitative Western blotting to determine the relative abundance of CC3, normalized to that of tubulin. Data are means ± SEM of two to five experiments per time point.

  • Table 1 MAPK pathway inhibitors produce differential apoptotic responses in the mouse intestinal epithelium.
    TreatmentpERK amountpMEK amountRelative apoptotic phenotypeCC3/tubulin AUC
    DMSOHighHighLate, high2.52
    PD325901LowHighEarly, high2.27
    SCH772984LowLowLate, low1.48
    MLN2480HighLowEarly, low1.03

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/407/ra129/DC1

    Fig. S1. Chemical structures of phosphoprotein signaling inhibitors.

    Fig. S2. MAPK pathway inhibitors alter the TNF-α–induced flux through multiple cellular signaling pathways.

    Fig. S3. D-PLSR analysis of the phosphoprotein signaling network.

    Fig. S4. SOM analysis of the phosphoprotein signaling network.

    Fig. S5. Influence of the Akt pathway inhibitors BEZ235 and MK-2206 on CC3 abundance and phosphoprotein signaling.

  • Supplementary Materials for:

    Network-level effects of kinase inhibitors modulate TNF-α–induced apoptosis in the intestinal epithelium

    Jessica J. Gierut, Levi B. Wood, Ken S. Lau, Yi-Jang Lin, Casie Genetti, Ahmed A. Samatar, Douglas A. Lauffenburger, Kevin M. Haigis*

    *Corresponding author. E-mail: khaigis{at}bidmc.harvard.edu

    This PDF file includes:

    • Fig. S1. Chemical structures of phosphoprotein signaling inhibitors.
    • Fig. S2. MAPK pathway inhibitors alter the TNF-α–induced flux through multiple cellular signaling pathways.
    • Fig. S3. D-PLSR analysis of the phosphoprotein signaling network.
    • Fig. S4. SOM analysis of the phosphoprotein signaling network.
    • Fig. S5. Influence of the Akt pathway inhibitors BEZ235 and MK-2206 on CC3 abundance and phosphoprotein signaling.

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    Citation: J. J. Gierut, L. B. Wood, K. S. Lau, Y.-J. Lin, C. Genetti, A. A. Samatar, D. A. Lauffenburger, K. M. Haigis, Network-level effects of kinase inhibitors modulate TNF-α–induced apoptosis in the intestinal epithelium. Sci. Signal. 8, ra129 (2015).

    © 2015 American Association for the Advancement of Science

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