The Unfolded Protein Response Plays a Predominant Homeostatic Role in Response to Mitochondrial Stress in Pancreatic Stellate Cells

Abstract

Activated pancreatic stellate cells (PaSC) are key participants in the stroma of pancreatic cancer, secreting extracellular matrix proteins and inflammatory mediators. Tumors are poorly vascularized, creating metabolic stress conditions in cancer and stromal cells that necessitate adaptive homeostatic cellular programs. Activation of autophagy and the endoplasmic reticulum unfolded protein response (UPR) have been described in hepatic stellate cells, but the role of these processes in PaSC responses to metabolic stress is unknown. We reported that the PI3K/mTOR pathway, which AMPK can regulate through multiple inputs, modulates PaSC activation and fibrogenic potential. Here, using primary and immortalized mouse PaSC, we assess the relative contributions of AMPK/mTOR signaling, autophagy and the UPR to cell fate responses during metabolic stress induced by mitochondrial dysfunction. The mitochondrial uncoupler rottlerin at low doses (0.5-2.5 μM) was added to cells cultured in 10% FBS complete media. Mitochondria rapidly depolarized, followed by altered mitochondrial dynamics and decreased cellular ATP levels. This mitochondrial dysfunction elicited rapid, sustained AMPK activation, mTOR pathway inhibition, and blockade of autophagic flux. Rottlerin treatment also induced rapid, sustained PERK/CHOP UPR signaling. Subsequently, high doses (>5 μM) induced loss of cell viability and cell death. Interestingly, AMPK knock-down using siRNA did not prevent rottlerin-induced mTOR inhibition, autophagy, or CHOP upregulation, suggesting that AMPK is dispensable for these responses. Moreover, CHOP genetic deletion, but not AMPK knock-down, prevented rottlerin-induced apoptosis and supported cell survival, suggesting that UPR signaling is a major modulator of cell fate in PaSC during metabolic stress. Further, short-term rottlerin treatment reduced both PaSC fibrogenic potential and IL-6 mRNA expression. In contrast, expression levels of the angiogenic factors HGF and VEGFα were unaffected, and the immune modulator IL-4 was markedly upregulated. These data imply that metabolic stress-induced PaSC reprogramming differentially modulates neighboring cells in the tumor microenvironment.

Fig 1. Rottlerin affects cellular metabolic state and induces apoptotic cell death.

Mouse PaSC were plated in 10% FBS medium and treated with DMSO as control or rottlerin at different concentrations for up to 72 h. (A) Levels of cellular metabolic activity measured by MTT assay in mPaSC treated with rottlerin for 72 h (control O.D values were set as 1). Graph represents the mean ±SEM; n = 3. (B) Apoptosis measured by internucleosomal DNA fragmentation in cell lysates and expressed relative to controls. Graph shows mean ± SEM; n = 4. (C) Secondary necrosis measured as internucleosomal DNA fragmentation in the conditioned media and expressed relative to controls. Graph shows mean ± SEM for 2 independent experiments. Asterisks in graphs indicate statistical significance (t-test, * p<0.05 as compared to control). (D) mPaSC were treated with 5 or 10 μM rottlerin for 24 hours. Cellular levels of the apoptotic regulators caspase-3 (pro-form and active cleaved-form), Bcl2, and Bim were measured by Western blotting. α-SMA was used as a marker of PaSC activation and as loading control; immunoblot is representative of two independent experiments.

Fig 2. Rottlerin induces rapid mitochondrial dysfunction in mPaSC that precedes LC3 puncta formation.

(A) mPaSC isolated from GFP-LC3 transgenic mice were labeled with 25 nM MitoTracker (MITO; red fluorescence) for 10 min and then treated without (control) or with 2.5 μM rottlerin for the indicated times. MITO staining (red) and GFP-LC3 puncta (green fluorescence) formation in live cells were visualized using a fluorescence microscope. (B) Rate of cellular oxygen consumption measured in live imPaSC under basal conditions and after sequential additions of DMSO and 1 μM rottlerin. Data is expressed as fold change relative to basal. Graph shows mean ± SEM; 3 independent experiments; * p<0.05 as compared to basal (t-test). (C) mPaSC were treated with 1 μM rottlerin for 30 or 60 minutes and total cellular ATP levels were measured in cell lysates by luminescence assay. Graph shows mean ± SEM for 3 independent experiments; * p<0.05 as compared to control (t-test). (D) mPaSC treated with 2.5 μM rottlerin for 1 or 24 hours were double immunostained for the mitochondrial marker Tom20 (green staining) and the stellate cell marker α-SMA (red staining); nuclei were counter-stained with DAPI (blue staining). Images were visualized under fluorescence microscope.

Fig 3. Rottlerin induces autophagy dysregulation in PaSC.

(A and B) Cells were treated with vehicle (-) or rottlerin for up to 48 h. Cellular protein levels of LC3-I and its LC3-phosphatidylethanolamine conjugate (LC3-II) were analyzed by Western blotting after short (A) and long-term treatment (B). Immunoblot in panel B also shows protein levels of the autophagy regulators p62/SQSTM1, LAMP-2, and Beclin-1. Fibronectin and α-SMA levels are shown as representative markers of stellate cell activation and loading controls. (C) Live imaging of GFP-LC3 mPaSC treated with vehicle or 2.5 μM rottlerin for up to 24 h by fluorescence microscopy. Representative pictures show diffuse cytoplasmic distribution of GFP-LC3 in control cells and GFP-LC3 puncta formation upon treatment with rottlerin. (D) mPaSC were immunostained for p62/SQSTM1 (green staining) and DAPI was used for visualization of nuclei. Pictures show progressive formation of p62 aggregates in mPaSC upon rottlerin treatment. (E and F) Blockade of autophagy by lysosomal inhibitors does not affect LC3 lipidation in mPaSC. Cells were pre-incubated for 30 min with bafilomycin A1 (E) or a lysosomal inhibitor cocktail (bafilomycin A1 + pepstatin A + E-64; panel F), and then incubated for 1 or 24 h with 1 μM rottlerin. Protein levels of LC3 and α-SMA were measured by Western blotting as autophagy marker and loading controls.

Fig 4. Rottlerin inhibits mTOR activation and autophagy in PaSC by AMPK-independent mechanisms.

(A) Primary mouse PaSC were treated with 1 μM rottlerin and Western blotting analysis was used to determine the activation state of AMPK and the mTORC1 targets p70 S6K and 4E-BP1. (B and C) Immortalized mouse PaSC were transfected with non-targeting (“con”) or AMPKα1/2 siRNA (“AMPK”) and then treated with rottlerin at the indicated concentrations for 1 h or 24 h. (B) Levels of total and phosphorylated AMPK, p70 S6K, S6 and ERK (loading control) were measured by Western blotting. (C) Levels of the autophagic markers LC3, p62, and GAPDH (loading control) were measured by Western blotting. (D) Cellular metabolic state of mock-transfected or AMPKα1/2 siRNA transfected imPaSC treated with vehicle or rottlerin for 72 hours was assessed by MTT assay. Graph shows O.D. values relative to control cells (mean ±SEM). As indicated in the graph, the reduction in cellular metabolic state induced by rottlerin was comparable in mock- and siRNA transfected cells. Data is representative of 3 independent experiments; ns = no statistical significant differences between control and AMPK siRNA transfected cells (t-test).

Fig 5. CHOP regulates cell death in rottlerin-treated PaSC.

(A and B) mPaSC were treated with different concentrations of rottlerin at the indicated times. Activation of the PERK/eIF2α branch of the UPR measured by the protein levels of total and phosphorylated eIF2α and the proapoptotic transcription factor CHOP. GAPDH expression was analyzed as a loading control. As shown, CHOP upregulation could be detected as early as 15 min in cells treated with 1 μM rottlerin (A) and this effect was sustained for at least 24 h (B). (C) Immunofluorescence for CHOP (red staining) in nuclei (blue DAPI staining) of mPaSC treated with 1μM rottlerin for 3 h and 24 h. (D) AMPKα1/2 were silenced by siRNA in imPaSC and then cells treated with rottlerin at indicated concentrations for 1 h or 24 h. p-eIF2α, total eIF2α, CHOP and GAPDH (loading control) were measured by Western blotting. (E) mPaSC isolated from wild type (WT) or Chop -/- mice were treated with rottlerin for 24 h. Immunoblots show protein levels of CHOP, GRP78, LC3 and GAPDH (loading control). (F and G) Cell death was assessed in WT or Chop -/- mPaSC treated with rottlerin for 48 h. Apoptosis was determined by DNA fragmentation ELISA (panel F) and cell number (panel G). Data in graphs is presented as mean ±SEM, n = 3; * p<0.05 as compared to WT; # p<0.05 as compared to control at 0 μM rottlerin (two way ANOVA followed by post-hoc Tukey tests). (H and I) p62 (panel H) and death receptor 5 (Dr5; panel I) mRNA levels were determined by qPCR in rottlerin-treated WT and Chop -/- mPaSC. As indicated, rottlerin-induced upregulation of p62 and Dr5 were blunted in Chop -/- cells. Data in graphs is presented as mean ±SEM, n = 3–4; * p<0.05 as compared to WT; # p<0.05 as compared to time 0 (two way ANOVA followed by post-hoc Tukey tests).

Fig 6. Metabolically stressed PaSC exhibit differential expression of profibrotic and inflammatory markers.

imPaSC were treated with rottlerin at different concentrations for 24 h. mRNA expression of (A) αSMA, (B) Col1a1, (C) Hgf, (D) Vegfα, (E) Il-6 and (F) Il-4 was determined by qPCR. Data in graph is mean ±SEM from three independent experiments; * p<0.05 as compared to WT (t-test).