Inhibition of 3-phosphoinositide-dependent protein kinase 1 (PDK1) can revert cellular senescence in human dermal fibroblasts

Abstract

Cellular senescence is defined as a stable, persistent arrest of cell proliferation. Here, we examine whether senescent cells can lose senescence hallmarks and reenter a reversible state of cell-cycle arrest (quiescence). We constructed a molecular regulatory network of cellular senescence based on previous experimental evidence. To infer the regulatory logic of the network, we performed phosphoprotein array experiments with normal human dermal fibroblasts and used the data to optimize the regulatory relationships between molecules with an evolutionary algorithm. From ensemble analysis of network models, we identified 3-phosphoinositide-dependent protein kinase 1 (PDK1) as a promising target for inhibitors to convert the senescent state to the quiescent state. We showed that inhibition of PDK1 in senescent human dermal fibroblasts eradicates senescence hallmarks and restores entry into the cell cycle by suppressing both nuclear factor κB and mTOR signaling, resulting in restored skin regeneration capacity. Our findings provide insight into a potential therapeutic strategy to treat age-related diseases associated with the accumulation of senescent cells.

Keywords: PDK1; cellular senescence; network modeling; skin aging; systems biology.

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.

Inference of multiple Boolean network models. (A) A schematic diagram of the cellular senescence network. The network consists of 41 nodes and 77 links: 48 links are activation links and 29 links are inhibition links. Not all links are direct; some intervening nodes are not included in the network. Shape and color define the 3 input nodes, 6 output nodes, and 32 internal nodes. Detailed information for each node and link is shown in SI Appendix, Table S1 and S2. (B) Hierarchical clustering heat map of phosphoprotein array data. AUC values of the indicated phosphorylated proteins were visualized using a hierarchical clustering heat map upon three conditions. (C) Heat map of binarized phosphoprotein arrays. Binarized AUC values of the indicated phosphorylated proteins were visualized using a heat map. (D) Average node activities of Boolean network models after the models reached their respective attractors. Input nodes and output nodes are highlighted by bold-lined boxes.

Fig. 2.

Ensemble model simulation to identify senescence-reversing targets. (A) Nodes were colored according to their scores indicating if inhibition resulted in a transition of the network output from senescence attractor to a proliferation attractor (blue, negative values) or from a senescence attractor to a quiescence attractor (red, positive values). The equation for determining the score is shown. (B) A bar graph of the ratio of Boolean network models for which inhibition of the listed node converts the senescence state into the quiescence state (R[node]sq). The values for IKBKB and CDKN2A are close to zero, but not zero. (C) A bar graph of the ratio of Boolean network models for which inhibition of the listed node converts the senescence state into the proliferation state (R[node]sp).

Fig. 3.

Mechanistic analysis of PDK1 inhibition as a method of converting the senescence state to the quiescent state. (A) Nodes were colored according to whether PDK1 inhibition increased node activity (red) or decreased activity (blue) using the value of E[node], which was calculated using the equation shown (see Materials and Methods for more details). Three nodes of mFVS are marked by yellow stars. The pill shape represents inhibition of PDK1. (B) Immunoblot showing the abundance of the indicated proteins or phosphorylated proteins in NHDFs. Young NHDFs were not subjected to senescence conditions or drug treatment. Samples from young NHDFs were collected when cultures reached ∼80% confluence at PDL 12 (Materials and Methods). The remaining samples were from NHDFs induced to senescence by treatment with doxorubicin (100 ng/mL) plus IGF1 (100 ng/mL) for 7 d. The senescent cells were then exposed to DMSO, rapamycin (100 nM), rapamycin plus JSH23 (2.5µM), or BX795 (100 nM) for 7 more days. Cell lysates were subjected to immunoblot analysis using antibodies against the indicated proteins. Data are representative of at least two independent experiments. (C) A diagram of the positive feedback loop that is deactivated by PDK1 inhibition. Each regulatory connection is labeled with its signal strength in the indicated condition (see Materials and Methods for details).

Fig. 4.

Senescence-associated phenotypes following PDK1 inhibition in NHDFs. Young NHDFs were not subjected to senescence conditions or drug treatment. Young NHDFs were analyzed when cultures reached ∼80% confluence at PDL 12. The remaining NHDFs were induced to senescence by 7-d exposure to doxorubicin (100 ng/mL) plus IGF1 (100 ng/mL). The senescent cells were then treated with DMSO, rapamycin (100 nM), rapamycin plus JSH (2.5µM), or BX795 (100 nM) for 7 more days. (A) Representative images of SA-β-gal staining. (Scale bars, 200 μm.) (B) Bar diagram showing percentage SA-β-gal–positive cells. (C) Representative immunofluorescent images of NHDFs stained for EdU (proliferating cells) and 53BP1 (cells with active DNA damage response). (Scale bars, 50 μm.) Last column shows a representative cell’s nucleus for each condition. (D) Quantification of EdU as percent of cells with positive staining. (E) Quantification of 53BP1-positive cells as percent of cells with positive nuclear staining. For B, D, and E, data are shown as mean ± SD (n = 5), ***P < 0.001 compared with the DMSO control senescent cells by one-way analysis of variance. ns, not statistically significant.

Fig. 5.

PDK1 inhibition on skin aging. Three-dimensional skin models (skin equivalents) were prepared from young NHDFs (PDL 12) or senescent NHDFs induced into senescence by doxorubicin plus IGF1 or repeated plating (replicative senescence, PDL 57) were exposed to DMSO or BX795. (A) Skin equivalent model sections were stained with hematoxylin and eosin (H&E) or Masson’s trichrome to detect collagen fibers. The indicated proteins were detected by immunohistochemistry. Results are from one representative experiment of three. (Scale bars, 50 µm.) (B) Quantification of dermal thickness using ImageJ software. Quantification of MMP1. Quantification of pan-collagen fiber detected by Masson’s trichrome staining. Quantification of Ki-67–positive cells. Quantification of epidermal thickness using ImageJ software. Quantification of keratin 10. Quantification of filaggrin. Quantification of COL17A1. Quantification of laminin5. Quantification of type I procollagen protein secreted into culture media quantified by ELISA. Data in B are shown as mean ± SD of three independent fields obtained from three independent samples. ***P < 0.001 compared to DMSO control senescent cells by one-way analysis of variance.