Modulation of the mevalonate pathway by akt regulates macrophage survival and development of pulmonary fibrosis

Abstract

Protein kinase B (Akt) is a key effector of multiple cellular processes, including cell survival. Akt, a serine/threonine kinase, is known to increase cell survival by regulation of the intrinsic pathway for apoptosis. In this study, we found that Akt modulated the mevalonate pathway, which is also linked to cell survival, by increasing Rho GTPase activation. Akt modulated the pathway by phosphorylating mevalonate diphosphate decarboxylase (MDD) at Ser(96). This phosphorylation in macrophages increased activation of Rac1, which enhanced macrophage survival because mutation of MDD (MDDS96A) induced apoptosis. Akt-mediated activation in macrophages was specific for Rac1 because Akt did not increase activity of other Rho GTP-binding proteins. The relationship between Akt and Rac1 was biologically relevant because Akt(+/-) mice had significantly less active Rac1 in alveolar macrophages, and macrophages from Akt(+/-) mice had an increase in active caspase-9 and -3. More importantly, Akt(+/-) mice were significantly protected from the development of pulmonary fibrosis, suggesting that macrophage survival is associated with the fibrotic phenotype. These observations for the first time suggest that Akt plays a critical role in the development and progression of pulmonary fibrosis by enhancing macrophage survival via modulation of the mevalonate pathway.

Keywords: Akt PKB; Apoptosis; Fibrosis; Macrophage; Oxidative Stress; Ras-related C3 Botulinum Toxin Substrate 1 (Rac1).

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

FIGURE 1.

Akt-deficient mice are protected from pulmonary fibrosis. WT and Akt+/− mice were exposed to 125 μg of chrysotile intratracheally. 21 days later, lungs from WT (A) and Akt+/− (B) mice were removed and processed for collagen deposition using Masson's trichome staining. Representative micrographs from 1 of 10 mice are shown. Bar, 100 μm for both A and B. C, hydroxyproline assay of lungs removed from WT and Akt+/− mice after chrysotile exposure (WT, n = 8; Akt+/−, n = 7). *, p = 0.043 versus WT. D, total number of BAL cells was counted in WT (n = 10) and Akt+/− (n = 10) mice after chrysotile exposure. *, p = 0.040 versus WT. E, cell differential was determined using Wright-Giemsa stain from BAL performed in WT and Akt+/− after chrysotile exposure (WT, n = 10; Akt+/−, n = 10). *, p < 0.001 versus polymorphonuclear (PMN) cells and lymphs. F, MCP-1 levels were measured in BAL fluid from WT (n = 9) and Akt+/− (n = 9) mice after chrysotile exposure. G, macrophages isolated from WT and Akt+/− mice were subjected to immunoblot analysis. H, measurement of alveolar macrophage H2O2 was performed in isolated mitochondria from BAL cells (WT, n = 6; Akt+/−, n = 6). *, p = 0.0118 versus Akt+/−. Lungs were removed and homogenized for the glutathione assay. I, reduced GSH levels in WT (n = 8) and Akt+/− (n = 8) mice. *, p = 0.019 versus WT. J, total GSH in disulfide form was expressed as the percentage of GSH as GSSG in WT (n = 9) and Akt+/− (n = 7) mice. *, p = 0.017 versus WT. Error bars, S.D.

FIGURE 2.

Akt specifically regulates Rac1 activity.A, macrophages were transiently transfected with either an empty or a constitutively active Akt (Akt) vector. Cells were incubated with 50 μm LY-294002 (LY) or vehicle (DMSO) for 60 min and exposed to chrysotile (10 μg/cm2) for 30 min. Whole cells were isolated, and immunoblot analysis was performed. Macrophages expressing Akt were analyzed for activation of Rac2 (B), RhoA (C), and Rac1 (D) using a pull-down assay and quantified by densitometry from three independent experiments. *, p < 0.001 versus empty. Inset, overexpression of Akt was confirmed by immunoblot analysis. E, macrophages were transfected with scrambled (Scr) or Akt siRNA, and Rac1 activity was determined as described above. Inset, knockdown of Akt was confirmed by immunoblot analysis. Macrophages expressing empty or Akt were incubated with 50 μm LY-294,200 hydrochloride or vehicle for 60 min. Mitochondrial (F) or cytoplasmic (G) fractions were isolated, and immunoblot analysis was performed. H, active Rac1 was determined in the mitochondrial fraction by the G-LISA kit in cells expressing empty vector or Akt (n = 9). *, p = 0.0132 versus empty; **, p < 0.0001 versus empty. Error bars, S.D.

FIGURE 3.

Akt phosphorylates MDD and regulates Rac1 geranylgeranylation.A, macrophages expressing empty or Akt vectors were incubated with 10 μm DGBP or vehicle (water) for 16 h and analyzed by immunoblot analysis for Rap 1A in whole cell lysates. Inset, overexpression of Akt was verified by immunoblot analysis. B, macrophages expressing empty vector or Akt were incubated with DGBP or vehicle and fractionated into aqueous (A) or detergent (D) phases. Lysates were subjected to immunoblot analysis. C, Rac1 activity was determined in macrophages overexpressing Rac1WT or Rac1CA and co-expressing empty vector or Akt. Macrophages were incubated with DGBP or vehicle (n = 3). *, p < 0.0001 versus Rac1WT (empty); **, p = 0.0029 versus Rac1WT (empty); ***, p < 0.0001 versus Rac1CA (empty); #, p < 0.0001 versus Rac1WT (empty); ##, p < 0.0001 versus Rac1CA (empty). D, Rac1 activity in macrophages overexpressing empty or Akt and treated with vehicle (ethanol) or 100 μm cholesterol for 24 h (n = 3). *, p < 0.0001 versus empty (vehicle); **, p = 0.0008 versus empty (vehicle). E, schematic diagram of the mevalonate pathway. Highlighted are MDD and DGBP, which inhibits geranylgeranyl diphosphate synthase (GGDPS). HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; MVADP, mevalonate 5-diphosphate; IDP, isopentenyl 5-diphosphate; FDP, farnesyl diphosphate; GGDP, geranylgeranyl diphosphate. F, macrophages transfected with either empty vector or Akt in combination with either empty vector or an MDD vector were subjected to SDS-PAGE (4–15% gradient gel). Immunoblot analysis for phospho-MDD and phospho-Akt was quantified by densitometry (n = 3). *, p < 0.0304 versus empty (empty); **, p = 0.0147 versus Akt (empty). G, cells expressing empty or Akt vectors were lysed in the presence or absence of phosphatase inhibitors. Lysates were subjected to SDS-PAGE (4–15% gradient gel) and immunoblot analysis. H, schematic diagram of full-length MDDWT construct and MDD constructs with mutations (Ser (S) → Ala (A)) at potential Akt phosphorylation sites. I, macrophages were transfected with empty or Akt vectors and either MDD-V5-HisWT, MDD-V5-HisS96A, MDD-V5-HisS231A, or MDD-V5-HisS96A,S231A. Lysates were subjected to His pull-down and immunoblot analysis for Ser(P). J, Rac1 activity was determined in macrophages expressing empty or MDD constructs using a pull-down assay (n = 4). *, p < 0.0001 versus empty; **, p < 0.0001 versus MDDWT (empty); ***, p < 0.0001 versus empty and versus MDDWT (empty); ****, p < 0.001 versus MDDS231A (empty). K, immunoblot analysis of macrophages transfected with either empty or Akt vectors in combination with either MDDWT or MDDS96A. L, immunoblot analysis of macrophages isolated from WT and Akt+/− mice. M, Rac1 activity in mitochondria isolated from BAL cells from WT (n = 6) and Akt+/− (n = 6) mice. *, p = 0.049 versus WT. Error bars, S.D.

FIGURE 4.

Rac1 is required for Akt-induced mitochondrial oxidative stress.A, macrophages were transfected with empty vector or Akt in combination with either empty or Rac1WT vectors. Mitochondria were isolated, and mitochondrial H2O2 was measured (n = 9). *, p < 0.0001 versus empty (empty); **, p < 0.0001 versus Akt (empty) and versus empty (Rac1WT). B, macrophages were transfected with scramble (Scr) or Rieske siRNA in combination with empty or Rac1WT vectors. Mitochondria were isolated, and mitochondrial H2O2 was measured. Inset, Rieske knockdown was verified by immunoblot analysis. n = 9. *, p < 0.0001 versus empty (scramble); **, p < 0.0001 versus empty (scramble). C, macrophages were transfected with scramble or Rac1 siRNA in combination with empty or Akt vectors. Mitochondria were isolated, and mitochondrial H2O2 was measured. Inset, Rac1 knockdown was verified by immunoblot analysis (n = 9). *, p < 0.001 versus empty (scramble); **, p < 0.0001 versus empty (scramble). D, macrophages were transfected with empty or Akt vectors and treated with catalase (PEG-CAT) for 1 h or pretreated with PEG-CAT and then exposed to chrysotile for 30 min (n = 8). *, p < 0.0001 versus empty; **, p < 0.001 versus Akt and versus empty (chrysotile); #, p < 0.0001 versus empty; ##, p < 0.02 versus empty (chrysotile). E, macrophages were transfected with empty or Rac1WT vectors and treated with catalase (PEG-CAT) or pretreated with PEG-CAT and then exposed to chrysotile (n = 8). *, p < 0.0001 versus empty; **, p < 0.001 versus Akt and versus empty (chrysotile); #, p < 0.0003 versus empty; ##, p < 0.001 versus empty (chrysotile). F, macrophages were transfected with empty or Akt vectors and either MDDWT or MDDS96A. H2O2 production was measured (n = 9). *, p = 0.031 versus empty (empty); **, p < 0.035 versus MDDWT (empty); ***, p < 0.004 versus empty (empty). G, macrophages were transfected with empty or Rac1WT vectors and either MDDWT or MDDS96A. H2O2 production was measured (n = 9). *, p < 0.014 versus empty (empty); **, p < 0.0027 versus Rac1WT (empty) and versus MDDWT (empty); ***, p < 0.004 versus empty (empty). H, macrophages were transfected with empty or Rac1CA vectors and either MDDWT or MDDS96A. H2O2 production was measured (n = 9). *, p < 0.0001 versus empty (empty); **, p < 0.0001 versus Rac1CA (empty) and versus MDDWT (empty); ***, p < 0.00014 versus empty (empty). Error bars, S.D.

FIGURE 5.

Akt mediates macrophage cell survival.A, representative images of apoptotic nuclear TUNEL staining in macrophages expressing empty and phMGFP-empty vectors or Akt and phMGFP-empty vectors. Cells were treated with 10 μm DGBP or vehicle for 16 h. B, quantitative analysis of A expressed as a ratio of number of TUNEL-positive and GFP-positive cells to the total number of GFP-positive cells. Counts were conducted on five different fields within each group. *, p < 0.001 versus empty and versus Akt. C, caspase-3 activity was determined in macrophages overexpressing Rac1WT or Rac1CA and co-expressing empty or Akt. Macrophages were incubated with DGBP or vehicle (n = 8). *, p < 0.0001 versus Rac1WT (Empty); **, p < 0.0001 versus Rac1CA (Empty); #, p < 0.0001 versus Rac1WT (Empty); ## p < 0.0001 versus Rac1CA (Empty). D, macrophages expressing Akt were analyzed for caspase-3 activity in cells incubated in vehicle (ethanol) or 100 μm cholesterol for 24 h. n = 8. *, p < 0.0001 versus empty (Vehicle); **, p = 0.0008 versus empty (Vehicle). E, caspase-3 activity was measured in macrophages expressing empty or Akt vectors and either MDDWT or MDDS96A. n = 8. *, p < 0.0001 versus empty (Empty); **, p < 0.0001 versus MDDWT (Empty); ***, p < 0.0001 versus MDDWT (Empty). F, macrophages were transfected with empty or Rac1WT vectors and treated with catalase (PEG-CAT) for 1 h or pretreated with PEG-CAT and then exposed to chrysotile for 30 min (n = 8). *, p < 0.0001 versus empty; **, p < 0.0425 versus Rac1WT (Vehicle) and versus empty (Chrysotile); #, p < 0.0376 versus empty; ##, p < 0.0174 versus empty. G, caspase-3 activity was analyzed in BAL cells from WT (n = 4) and Akt+/− (n = 4) mice. *, p < 0.0406 versus WT. Error bars, S.D.

FIGURE 6.

Akt modulates M2 macrophage polarization and TGF-β1 gene expression.A, total RNA was isolated, and mRNA expression was measured by real-time PCR in macrophages expressing empty or Akt vectors. Results show arbitrary units normalized to β-actin mRNA (n = 9). *, p = 0.0011 versus IL-1β empty; **, p = 0.0036 versus Arg I empty; ***, p = 0.0027 versus Ym1 empty; ****, p = 0.0015 versus TGF-β1 empty. B, TGF-β mRNA expression was measured in macrophages expressing empty or Rac1WT together with empty or Akt. Results show arbitrary units normalized to hypoxanthine-guanine phosphoribosyltransferase mRNA (n = 9). *, p = 0.0012 versus empty (empty); **, p = 0.0495 versus empty (empty with chrysotile); ***, p = 0.0381 versus Rac1 (empty); ****, p = 0.0381 versus Rac1 (empty with chrysotile). C, human lung fibroblasts (HLF-1) were incubated with conditioned medium from macrophages expressing empty or Akt vectors. Shown is immunoblot analysis of procollagen and collagen I from medium. The graph shows densitometry from three independent experiments (n = 9). *, p = 0.009 versus empty. D, HLF-1 were incubated with conditioned medium from macrophages expressing scramble (Scr) or Akt siRNA. Knockdown of Akt was confirmed by immunoblot analysis (inset). Quantification of collagen I expression from three separate experiments by densitometry (n = 9). *, p = 0.022 versus scramble. E, mouse lung fibroblasts from WT mice were incubated with BAL fluid from chrysotile exposed WT (n = 5) or Akt+/− (n = 5) mice. Immunoblot analysis of procollagen and collagen I from medium, which was quantified by densitometry. *, p = 0.015 versus WT. F, arginase I mRNA expression was measured in macrophages expressing empty or Akt together with MDDWT or MDDS96A. Results show arbitrary units normalized to β-actin mRNA. n = 9. *, p = 0.0132 versus empty (empty); **, p = 0.0156 versus MDDWT (empty). G, TNF-α, Ym1, and active TGF-β1 levels were measured in BAL fluid from WT (n = 9) and Akt+/− (n = 10) mice 21 days after chrysotile exposure. *, p = 0.0031 versus Ym1 WT; **, p = 0.0039 versus TGF-β1 WT. Error bars, S.D.

FIGURE 7.

Alveolar macrophages from asbestosis patients have increased Akt activation and cell survival. Alveolar macrophages were obtained from normal subjects and asbestosis patients. A, a representative immunoblot analysis for phospho-Akt in whole cell lysates is shown. A quantitative analysis of phospho-Akt immunoblots normalized to total Akt is shown by densitometry in normal subjects (n = 4) and asbestosis patients (n = 4). *, p = 0.0048 versus normal. B, a representative immunoblot analysis for phospho-MDD in whole cell lysates subjected to SDS-PAGE (4–15% gradient gel) is shown. A quantitative analysis of phospho-MDD immunoblots normalized to total MDD by densitometry is shown in normal subjects (n = 4) and asbestosis patients (n = 4). *, p = 0.014 versus normal. C, activity of Rac1, Rac2, and RhoA was measured in whole cell lysates of alveolar macrophages from normal subjects (n = 4) and asbestosis patients (n = 4) using a pull-down assay. Immunoblots were quantified by densitometry of each GTPase protein relative to GST expression. *, p = 0.0221 versus Rac1 normal subjects. **, p = 0.0132 versus Rac2 normal subjects. D, immunoblot analysis from normal subjects (n = 4) and asbestosis patients (n = 5) was quantified by densitometry for active caspase-9 and -3 relative to β-actin. *, p = 0.0268 versus caspase-9 normal subjects. **, p = 0.0148 versus caspase-3 normal subjects. Error bars, S.D.