Cystathionine β-synthase and cystathionine γ-lyase double gene transfer ameliorate homocysteine-mediated mesangial inflammation through hydrogen sulfide generation

Abstract

Elevated level of homocysteine (Hcy) induces chronic inflammation in vascular bed, including glomerulus, and promotes glomerulosclerosis. In this study we investigated in vitro mechanism of Hcy-mediated monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) induction and determined the regulatory role of hydrogen sulfide (H₂S) to ameliorate inflammation. Mouse glomerular mesangial cells (MCs) were incubated with Hcy (75 μM) and supplemented with vehicle or with H₂S (30 μM, in the form of NaHS). Inflammatory molecules MCP-1 and MIP-2 were measured by ELISA. Cellular capability to generate H₂S was measured by colorimetric chemical method. To enhance endogenous production of H₂S and better clearance of Hcy, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) genes were delivered to the cells. Oxidative NAD(P)H p47(phox) was measured by Western blot analysis and immunostaining. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH₂-terminal kinase (JNK1/2) were measured by Western blot analysis. Our results demonstrated that Hcy upregulated inflammatory molecules MCP-1 and MIP-2, whereas endogenous production of H₂S was attenuated. H₂S treatment as well as CBS and CSE doubly cDNA overexpression markedly reduced Hcy-induced upregulation of MCP-1 and MIP-2. Hcy-induced upregulation of oxidative p47(phox) was attenuated by H₂S supplementation and CBS/CSE overexpression as well. In addition to that we also detected Hcy-induced MCP-1 and MIP-2 induction was through phosphorylation of ERK1/2 and JNK1/2. Either H₂S supplementation or CBS and CSE doubly cDNA overexpression attenuated Hcy-induced phosphorylation of these two signaling molecules and diminished MCP-1 and MIP-2 expressions. Similar results were obtained by inhibition of ERK1/2 and JNK1/2 using pharmacological and small interferring RNA (siRNA) blockers. We conclude that H₂S plays a regulatory role in Hcy-induced mesangial inflammation and that ERK1/2 and JNK1/2 are two signaling pathways involved this process.

Fig. 1.

A: homocysteine (Hcy) dose dependently induced monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) in mouse glomerular mesangial cells (MCs). MCs were plated onto 12-well cell culture plate as described in the materials and methods. Cells were treated with l-Hcy (0–100 μM) for 48 h as shown in the figure. Supernatant was collected, and cells were lysed with RIPA lysis buffer. MCP-1 was measured in cell lysates, and MIP-2 was measured in supernatant by ELISA kit as described in materials and methods. Detected levels of MCP-1 and MIP-2 in the l-Hcy-treated groups were expressed as percentage of control. In a separate set of experiment, MCs were also treated with methionine (Met, 75 μM) and l-cysteine (Cys, 75 μM) for 48 h, and MCP-1 and MIP-2 were measured and expressed as percent control (inset). Data represents means ± SE; n = 5 independent experiments, *P < 0.01 vs. control. B: Hcy-mediated attenuation of H₂S was improved by cystathionine β-synthase (CBS) and cystathione γ-lyase (CSE) gene delivery. MCs were treated with Met (75 μM), l-Cys (75 μM), and l-Hcy (75 μM, with or without CBS/CSE/double gene transfection) for 48 h, and the cell capabilities to generate H₂S were measured by previously adopted method (39). Data represent means ± SE; n = 5 independent experiments. *P < 0.025 vs. control, #P < 0.05 vs. control. C: expression of CBS/CSE genes in MCs. MCs were transfected with pcDNA3.1/GFP, pcDNA3.1/CBS, pME18S-CSE-HA, or both CBS and CSE DNA as described (38). After 48 h, the transfection efficacy was determined by examining pcDNA/GFP-transfected cells for green fluorescent protein (GFP) fluorescence using fluorescent microscope (Olympus IX51). Approximately 50% cells were found positive (original magnification, ×40). The CBS- and CSE-transfected cells had no fluorescence activity. Expression of CBS (D) and CSE (E) protein were determined by Western blot analysis.

Fig. 2.

Increased ability of endogenous H₂S production or exogenous supplementation attenuated Hcy-mediated MCP-1 and MIP-2 induction in MCs. MCs were transfected with CBS, CSE, or both cDNAs as described (38) or cultured without any transfection. Cells were treated with l-Hcy (75 μM) and supplemented with or without H₂S (in the form of NaHS solution) in appropriate groups as shown in the figure. MCP-1 and MIP-2 were measured as described in the Fig. 1. Data represent means ± SE; n = 5 independent experiments. *P < 0.01 vs. control and †P < 0.05 vs. Hcy.

Fig. 3.

CBS and CSE double gene therapy attenuated Hcy-induced p47^phox upregulation in MCs. A: MCs were cultured in 8-well chamber slide and transiently transfected with CBS, CSE, or both the cDNAs and treated with Hcy (75 μM) for 48 h. Cells were fixed, permeabilized, blocked with BSA in PBS, and immunostained with anti-p47^phox antibody secondarily conjugated with Texas Red. Cells were also counterstained with DAPI. Fluorescence images were taken under laser scanning confocal microscope (Fluoview 1000, Olympus) and merged. Red fluorescence indicates p47^phox expression. Representative images from four independent experiments were shown here. B: similar experiment was performed in 12-well plastic plates and expression of p47^phox protein was determined by Western blot analysis. Blots reprobed with β-actin for loading control. C: bar diagram showing relative expression of p47^phox normalized with β-actin loading control. Data represent means ± SE; n = 4. *P < 0.01 vs. control; ϕP < 0.05 vs. Hcy.

Fig. 4.

H₂S inhibited extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH₂-terminal kinase (JNK1/2) phosphorylation induced by Hcy. A: ERK1/2 was either inhibited by small interferring RNA (siRNA) or blocked with pharmacological inhibitor (PD98059, 50 μM) or supplemented with H₂S (30 μM, in the form of NaHS) and treated with Hcy (75 μM) for 30 min. Appropriate controls were taken. Cells were lysed in RIPA lysis buffer and phosphorylation of ERK1/2 was detected by Western blot analysis. The blot reprobed with β-actin as loading control. B: bar diagram showed relative changes of phosphorylation over control (p-ERK1/2/β-actin). Data represent means ± SE; n = 5 independent experiments. *P < 0.05 vs. control; †P < 0.05 vs. Hcy treatment. C: in a similar set of experiments, JNK1/2 was either inhibited by siRNA or blocked with pharmacological inhibitor (SP600125, 10 μM) or supplemented with H₂S (30 μM) and treated with Hcy (75 μM) for 30 min with appropriate controls. Phosphorylation of JNK1/2 was detected by Western blot analysis. Blot reprobed with β-actin as loading control. D: bar diagram showed the relative changes of phosphorylation over control (p-JNK1/2/β-actin). Data represents means ± SE; n = 5 independent experiments. *P < 0.05 vs. control; †P < 0.05 vs. Hcy treatment.

Fig. 5.

CBS and CSE double gene transfection attenuated Hcy-induced ERK1/2 and JNK1/2 phosphorylation in MCs. A: MCs were transfected with CBS, CSE, or both the genes, and after 48 h of transfection, cells were treated with Hcy (75 μM) for 30 min. Appropriate controls were taken. Cells were lysed in RIPA lysis buffer, and phosphorylation of ERK1/2 was detected by Western blot analysis. The blot reprobed with β-actin as loading control. B: bar diagram showed relative changes of ERK1/2 phosphorylation over control (p-ERK1/2/β-actin). Data represents means ± SE; n = 5 independent experiments. *P < 0.05 vs. control; †P < 0.05 vs. Hcy treatment. C: in a similar set of experiment, JNK1/2 phosphorylation was detected by Western blot analysis. Blots were reprobed with β-actin as loading control. D: bar diagram showed the relative changes of phosphorylation over control (p-JNK1/2/β-actin). Data represent means ± SE; n = 5 independent experiments. *P < 0.05 vs. control; †P < 0.05 vs. Hcy treatment.

Fig. 6.

CBS and CSE through H₂S generation inhibited Hcy-induced MCP-1 and MIP-2 induction via ERK1/2 and JNK1/2 pathways. MCs were transfected with CBS, CSE, or double genes. In appropriate wells, as shown in the figure, ERK1/2 and JNK1/2 were inhibited by pharmacological blockers and treated with Hcy (75 μM) for 48 h. As showed in the figure, some wells also treated with Hcy (75 μM for 48 h) with or without H₂S. At the end of experiments, supernatant was collected, and cells were lysed with RIPA lysis buffer. MCP-1 and MIP-2 were measured as described in materials and methods. Data represent means ± SE; n = 5 independent experiments. *P < 0.01 vs. control and †P < 0.05 vs. Hcy.

Fig. 7.

Schematic of homocysteine-mediated signaling pathways leading to MCP-1 and MIP-2 expressions in mouse MCs. Hcy activates NADPH oxidase p47^phox subunit resulting in increased generation of O₂^·− (superoxide), which phosphorylates ERK1/2 and JNK1/2. Activation of these signaling cascades induces MCP-1 and MIP-2 expressions. Hcy also competitively inhibit CBS/CSE enzymes leading to decrease endogenous generation of H₂S. H₂S is an antioxidant, and its reduction results further increase of oxidative stress that amplify Hcy-mediated MCP-1 and MIP-2 expression. CBS/CSE gene delivery enhances cellular ability to produce H₂S from Hcy and attenuates Hcy-mediated MCP-1 and MIP-2 expression resulting in amelioration of inflammatory response in MCs.