TNF-related apoptosis-inducing ligand promotes human preadipocyte proliferation via ERK1/2 activation

Abstract

Upon obesity, adipose tissue is excessively expanded and characterized by pathologic processes like hypoxia, fibrosis, and inflammation. Death ligands belonging to the TNF superfamily such as TNF-α are important contributors to these derangements and exert a pronounced influence on the metabolic and cellular homeostasis of adipose tissue. Here, we sought to identify the effect of the death ligand TNF-related apoptosis-inducing ligand (TRAIL) on the adipose tissue precursor cell pool and therefore investigated its influence on preadipocyte proliferation. Treatment of human preadipocytes with TRAIL resulted in a time- and dose-dependent increase in proliferation (EC50 3.4 ng/ml) comparable to IGF-1. Although no apoptosis was observed, TRAIL triggered a rapid cleavage of caspase-8 and -3. Neither inhibition of caspase activity by zVAD.fmk (20 µM) nor ablation of caspase-8 expression by lentivirus-delivered small hairpin RNA (shRNA) abolished the proliferative response. TRAIL triggered a delayed and sustained activation of ERK1/2, leaving Akt, p38, JNK, and NF-κB unaffected. Importantly, inhibition of ERK1/2 activation by PD0325901 (300 nM) or AZD6244 (5 or 10 µM) completely abolished the proliferative response. We thus reveal a hitherto unknown function of TRAIL in regulating adipose tissue homeostasis by promoting the proliferation of tissue-resident precursor cells.

Keywords: adipocyte progenitor; adipose tissue homeostasis; death ligand; noncanonical signaling.

Figure 1.

TRAIL stimulates human preadipocyte proliferation. A–C) SGBS preadipocytes were stimulated with growth medium alone or with increasing doses of TRAIL. A) At the indicated time points, the number of adherent cells was counted using a net micrometer. Data are expressed as means ± sem of 3 independent experiments performed. *P ≤ 0.05 for TRAIL 100 ng/ml compared with growth medium alone at 72 hours. B) After 72 hours, [³H]-thymidine incorporation was measured. Data are expressed as means ± sem of at least 3 independent experiments performed. **P ≤ 0.01; ***P ≤ 0.001 compared with growth medium alone. C) After 72 hours, Hoechst 33342 fluorescence staining was performed. One representative of 3 independent experiments performed is shown. Scale bars, 200 µm. D) SGBS preadipocytes were stained with CFSE and stimulated with basal medium, growth medium alone or with TRAIL. After 72 hours, the remaining CFSE fluorescence was measured. A representative histogram of 1 of 3 independent experiments performed is shown. The loss of CFSE mean fluorescence intensity in comparison to cells stimulated with basal medium was calculated. *P ≤ 0.05 for TRAIL 30 ng/ml compared with growth medium alone. E) Human primary stromal-vascular cells were isolated from adipose tissue samples of 4 patients and stimulated with growth medium alone or with increasing doses of TRAIL. After 72 hours, [³H]-thymidine incorporation was measured. Data are expressed as means ± sem. *P ≤ 0.05; **P ≤ 0.01 compared with growth medium alone. F) SGBS preadipocytes were stimulated with growth medium alone or human agonistic antibodies specific for either TRAIL-R1 (mapatumumab, mapa) or TRAIL-R2 (lexatumumab, lexa). After 72 hours, [³H]-thymidine incorporation was measured. Data are expressed as means ± sem of 3 independent experiments performed.

Figure 2.

TRAIL does not induce human preadipocyte apoptosis. A, B) SGBS preadipocytes were stimulated with growth medium alone or with increasing doses of TRAIL in the absence or presence of cycloheximide (CHX). After 48 hours, apoptotic cell death was measured by flow cytometry. A) Representative histograms of 1 of 3 independent experiments performed are shown. B) Specific apoptosis was calculated and analyzed statistically. Data are expressed as means ± sem of 3 independent experiments performed. ***P ≤ 0.001 compared with growth medium alone.

Figure 3.

Death ligands stimulate human preadipocyte proliferation. A) SGBS preadipocytes were stimulated with growth medium alone or with increasing doses of either FasL or TNF-α. After 72 hours, [³H]-thymidine incorporation was measured. Data are expressed as means ± sem of 3 independent experiments performed. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 compared with growth medium alone. B) SGBS preadipocytes were stimulated with growth medium alone or with either TRAIL or increasing doses of IGF-1. After 72 hours, [³H]-thymidine incorporation was measured. Data are expressed as means ± sem of at least 3 independent experiments performed. ***P ≤ 0.001 compared with growth medium alone.

Figure 4.

TRAIL triggers caspase activation, but caspases are not involved in the induction of proliferation. A) SGBS preadipocytes were stimulated with growth medium alone or with TRAIL. At the indicated time points, proteins were isolated and subjected to Western blot analysis to assess the processing of caspase-8 and -3. β-actin was used as a loading control. One representative of 3 independent experiments performed is shown. B, C) SGBS preadipocytes were stimulated with growth medium alone or with TRAIL and/or zVAD.fmk. B) At the indicated time points, proteins were isolated and subjected to Western blot analysis to assess the inhibition of caspase-8 and -3 processing by zVAD.fmk. β-actin was used as a loading control. One representative of 3 independent experiments performed is shown. C) After 72 hours, [³H]-thymidine incorporation was measured. Data are expressed as means ± sem of six independent experiments performed. *P ≤ 0.05; ***P ≤ 0.001 compared with growth medium alone (directly above the bars). ns, not significant comparing the indicated treatments (above the connecting line). D, E) A stable, lentiviral-mediated knockdown of caspase-8 in SGBS preadipocytes was performed. Two shRNAs targeting caspase-8 (shC8.1 and shC8.2) were used. A hyper random sequence (shHRS) was used as a control. Subsequent experiments were performed with cells from bulk cultures. D) Western blot analysis to assess the extent of caspase-8 knockdown by shRNA expression. α-tubulin was used as a loading control. E) Stably modified SGBS preadipocytes were stimulated with growth medium alone or with increasing doses of TRAIL. After 72 hours, [³H]-thymidine incorporation was measured. Data are expressed as means ± sem of 3 independent experiments performed. **P ≤ 0.01; ***P ≤ 0.001 compared with growth medium alone.

Figure 5.

TRAIL does not alter Akt or NF-κB signaling. A) SGBS preadipocytes were stimulated with growth medium alone or with TRAIL. At the indicated time points, proteins were isolated and subjected to Western blot analysis to assess the activation state of the Akt and NF-κB pathways. β-actin was used as a loading control. “C” indicates a positive control. One representative of 3 independent experiments performed is shown. B, C) SGBS preadipocytes were stimulated with growth medium alone or with either TRAIL or TNF-α. B) At the indicated time points, NF-κB p65 immunofluorescence staining was performed. One representative of 3 independent experiments performed is shown. Scale bars, 100 µm. C) At the indicated time points, nuclear proteins were isolated and subjected to EMSA analysis to assess the transcriptional activity of NF-κB. “P” indicates a control for the probe alone. Asterisk indicates nonspecific signals. One representative of 3 independent experiments performed is shown.

Figure 6.

TRAIL-mediated activation of ERK1/2 is responsible for the induction of proliferation. A) SGBS preadipocytes were stimulated with growth medium alone or with TRAIL. At the indicated time points, proteins were isolated and subjected to Western blot analysis to assess the activation state of the ERK1/2, p38, and JNK pathways. β-actin was used as a loading control. “C” indicates a positive control. One representative of 3 independent experiments performed is shown. B, C) SGBS preadipocytes were stimulated with growth medium alone or with TRAIL in the absence or presence of different doses of PD0325901 or AZD6244. B) At the indicated time points, proteins were isolated and subjected to Western blot analysis to assess the inhibition of ERK1/2 activation by PD0325901 or AZD6244. α-tubulin was used as a loading control. One representative of 3 independent experiments performed is shown. C) After 72 hours, [³H]-thymidine incorporation was measured. Data are expressed as means ± sem of 3 independent experiments performed. **P ≤ 0.01; ***P ≤ 0.001 compared with growth medium alone (directly above the bars) or comparing the indicated treatments (above the connecting line). D) SGBS preadipocytes were stimulated with growth medium alone or with TRAIL in the absence or presence of cycloheximide (CHX) or different doses of PD0325901 or AZD6244. After 48 hours, apoptotic cell death was measured by flow cytometry and specific apoptosis was calculated. Data are expressed as means ± sem of 3 independent experiments performed. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 compared with growth medium alone (directly above the bars) or comparing the indicated treatments (above the connecting line).