Redox and mTOR-dependent regulation of plasma lamellar calcium influx controls the senescence-associated secretory phenotype

Abstract

Through its ability to evoke responses from cells in a paracrine fashion, the senescence-associated secretory phenotype (SASP) has been linked to numerous age-associated disease pathologies including tumor invasion, cardiovascular dysfunction, neuroinflammation, osteoarthritis, and renal disease. Strategies which limit the amplitude and duration of SASP serve to delay age-related degenerative decline. Here we demonstrate that the SASP regulation is linked to shifts in intracellular Ca²⁺ homeostasis and strategies which rescue redox-dependent calcium entry including enzymatic H₂O₂ scavenging, TRP modulation, or mTOR inhibition block SASP and TRPC6 gene expression. As Ca²⁺ is indispensable for secretion from both secretory and non-secretory cells, it is exciting to speculate that the expression of plasma lamellar TRP channels critical for the maintenance of intracellular Ca²⁺ homeostasis may be coordinately regulated with the SASP.

Keywords: SASP; Senescence; TRPC6; calcium; hydrogen peroxide; mTOR.

Figure 1.

Senescence is accompanied by dysregulation of intracellular Ca²⁺ signaling mechanisms. (a) Pre-senescent (<p15) and senescent (>p25) levels of intracellular Ca²⁺ as measured using the inverse Ca²⁺ fluorescent dye Fura Red as monitored by imaging flow cytometer under ambient (21%) and low (3%) oxygen conditions. 5000 individual cells (events) were imaged. (b) Quantification of mean Fura Red relative fluorescence intensity representing the RFU ± SEM of multiple independent experiments, n = 5000 events. (c) Representative ratiometric Fura-2 Ca²⁺ imaging trace of pre-senescent (<p15) and senescent (>p25) fibroblasts treated with an exogenous source of Ca²⁺ (2 mM). At time 40 s bath media was changed to include 2 mM Ca²⁺. (d) Total change in intracellular Ca²⁺ after addition of 2 mM Ca²⁺ stimulus represented as ΔFura 340/380 ± SEM, n = 3. (e) Ratiometric Ca²⁺ imaging of pre-senescent and senescent fibroblasts untreated or treated with 10 μM SKF-96365 15 min before imaging. At time 40 s media was changed to include 2 mM Ca²⁺. (f) Total change in intracellular Ca²⁺ after addition of 2 mM Ca²⁺ stimulus represented as ΔFura 340/380 ± SEM, n = 3. *P ≤0.05, **P <0.01, ***P <0.001, ^#P < 0.0001. (A color version of this figure is available in the online journal.)

Figure 2.

Senescence-associated disruption of Ca²⁺ homeostasis is H₂O₂-regulated. (a) Ratiometric Ca²⁺ imaging of pre-senescent (<p15) and senescent (>p25) fibroblasts with 2 mM Ca²⁺ added at time 330 s and stimulated with increasing H₂O₂ concentrations of 0 μM, 50 μM, 150 μM, and 300 μM. (b) Quantification of total change in intracellular Ca²⁺ after addition of 2 mM Ca²⁺ stimulus with varying H₂O₂ concentrations represented as ΔFura 340/380 ± SEM, n = 3. (c) Ratiometric Ca²⁺ imaging of pre-senescent (<p15) and senescent fibroblasts pre-incubated with 200 U/mL recombinant catalase overnight with 2 mM Ca²⁺ added at time 330 s and stimulated with increasing H₂O₂ concentrations of 0 μM, 50 μM, 150 μM, and 300 μM. (d) Quantification of total change in intracellular Ca²⁺ after addition of 2 mM Ca²⁺ stimulus with varying H₂O₂ concentrations represented as ΔFura 340/380 as shown in Figure 1. (A color version of this figure is available in the online journal.)

Figure 3.

TRPC6 is overexpressed during senescence and its agonist unresponsiveness is restored by catalase pretreatment. Relative mRNA levels of TRPC3 and TRPC6 channels in pre-senescent (p25) cells. (b) TRPC6 and GAPDH protein abundance as measured from cell lysates of pre-senescent and senescent cells. (c) Ratiometric Ca²⁺ imaging of pre-senescent and senescent fibroblasts with 10 μM hyperforin and 2 mM Ca²⁺ added at time 60 s. (d) Quantification of total change in intracellular Ca²⁺ after addition of 10 μM hyperforin and 2 mM Ca²⁺ stimulus represented as ΔFura 340/380 ± SEM, n = 3. (e) Ratiometric Ca²⁺ imaging of pre-senescent and senescent fibroblasts pre-incubated with 200 U/mL recombinant catalase overnight with 10 μM hyperforin and 2 mM Ca²⁺ added at time 60 s (f) Quantification of total change in intracellular Ca²⁺ after addition of 10 μM hyperforin and 2 mM Ca²⁺ stimulus represented as shown in Figure 1. (A color version of this figure is available in the online journal.)

Figure 4.

SKF reverses senescence Ca²⁺ dysregulation and suppresses SASP. (a) Ratiometric Ca²⁺ imaging of senescent fibroblasts with 2 mM Ca²⁺ added at time 40 s following 15 min treatment with or without 10 μM SKF-96365 for and stimulated with increasing H₂O₂ concentrations of 0 μM, 50 μM, 150 μM, and 300 μM. (b) Total change in intracellular Ca²⁺ after addition of 2 mM Ca²⁺ stimulus with varying H₂O₂ concentrations of 0 μM, 50 μM, 150 μM, and 300 μM as shown in Figure 1. (c) Relative mRNA levels of key senescence and SASP components in pre-senescent and senescent cells as measured by real-time PCR. Results expressed as mean ± SEM and statistically compared against >p25 NT, n = 3. *P ≤0.05, ^#P <0.0001. (A color version of this figure is available in the online journal.)

Figure 5.

mTOR enhances Ca²⁺ entry and limits replicative SASP and TRPC6 expression. Ratiometric Ca²⁺ imaging of senescent fibroblasts with 2 mM Ca²⁺ added at time 40 s previously untreated or treated with 25 nM rapamycin or 10 μM Ku0063794 and stimulated with increasing H₂O₂ concentrations of 0 μM, 50 μM, 150 μM, and 300 μM. (b) Total change in intracellular Ca²⁺ after addition of 2 mM Ca²⁺ stimulus with varying H₂O₂ concentrations of 0 μM, 50 μM, 150 μM, and 300 μM as shown in Figure 1. (c) Relative mRNA levels of key senescence and SASP components in untreated fibroblasts and senescent (>p25) fibroblasts pre-incubated with 25 nM rapamycin or 10 μM Ku0063794 for 24 has measured by real-time PCR. Results expressed as mean ± SEM and statistically compared against >p25 NT, n = 3. *P ≤0.05, **P <0.01, ^#P <0.0001. (A color version of this figure is available in the online journal.)