Regulation of Cardiac Fibroblast GLS1 Expression by Scleraxis

Abstract

Fibrosis is an energy-intensive process requiring the activation of fibroblasts to myofibroblasts, resulting in the increased synthesis of extracellular matrix proteins. Little is known about the transcriptional control of energy metabolism in cardiac fibroblast activation, but glutaminolysis has been implicated in liver and lung fibrosis. Here we explored how pro-fibrotic TGFβ and its effector scleraxis, which drive cardiac fibroblast activation, regulate genes involved in glutaminolysis, particularly the rate-limiting enzyme glutaminase (GLS1). The GLS1 inhibitor CB-839 attenuated TGFβ-induced fibroblast activation. Cardiac fibroblast activation to myofibroblasts by scleraxis overexpression increased glutaminolysis gene expression, including GLS1, while cardiac fibroblasts from scleraxis-null mice showed reduced expression. TGFβ induced GLS1 expression and increased intracellular glutamine and glutamate levels, indicative of increased glutaminolysis, but in scleraxis knockout cells, these measures were attenuated, and the response to TGFβ was lost. The knockdown of scleraxis in activated cardiac fibroblasts reduced GLS1 expression by 75%. Scleraxis transactivated the human GLS1 promoter in luciferase reporter assays, and this effect was dependent on a key scleraxis-binding E-box motif. These results implicate scleraxis-mediated GLS1 expression as a key regulator of glutaminolysis in cardiac fibroblast activation, and blocking scleraxis in this process may provide a means of starving fibroblasts of the energy required for fibrosis.

Keywords: cardiac fibrosis; energy metabolism; fibroblast; gene regulation; glutaminolysis; myofibroblast; transcription.

Figure 1

TGFβ-induced fibroblast activation and GLS1 expression is attenuated by CB-839. (A–F) Rat cardiac fibroblasts were treated for 24 h with vehicle (V) or 10 ng/mL TGFβ₁, with or without GLS1 inhibitor CB-839 (CB, 0.3 μM), then assayed for expression of periostin (Postn) mRNA by qPCR (A), secretion of Postn (B), development of stress fibers (C), proliferation (D), and expression of GLS1 (E) and Acot2 mRNA by qPCR (F). In a similar experiment, protein expression of GLS1 and aldolase C (ALDOC) was assessed by Western blot (G). Statistical significance was determined by one-way ANOVA with the Tukey post hoc test (n = 3–4) (A,B,E–G), or by the Kruskal–Wallis test followed by Dunn’s multiple comparisons test (D, n = 16–18; ALDOC in G, n = 4); images in (C) are representative of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus vehicle; # p < 0.05, #### p < 0.0001 versus TGFβ. Scale bar in (C), 50 μm.

Figure 2

Fibroblast activation and scleraxis overexpression upregulate glutaminolysis gene expression. (A,B) Non-passaged (P0), freshly-isolated rat cardiac fibroblasts, or twice-passaged P2 rat cardiac myofibroblasts were assayed for mRNA expression of scleraxis (A) and GLS1 (B) by qPCR. (C–G) P1 rat cardiac fibroblasts were transfected by adenovirus encoding green fluorescent protein (GFP), or scleraxis (Scx) for 48 h, then assayed by qPCR for expression of GLS1 (C), GLS2 (D), GOT2 (E), and GDH1 (F), or were analyzed by Western blot for expression of GLS1, ALDOC, and Acot2 (G). Statistical analysis was by the two-tailed Student’s t-test (n = 3–4). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus P0 or GFP.

Figure 3

Loss of scleraxis attenuates glutaminolysis gene expression. (A,B) Cardiac fibroblasts were isolated from wild-type (WT) or scleraxis knockout (KO) mice and assayed for the expression of GLS1 (A) and GDH1 (B) by qPCR. (C,D) P1 rat cardiac fibroblasts were transfected with adenovirus encoding small hairpin RNA targeting LacZ (AdshLacZ) or scleraxis (AdshScx) for 72 h, with or without 10 ng/mL TGFβ₁ addition after 48 h, and assayed by qPCR for expression of scleraxis (C) and GLS1 (D). Statistical analysis was by the two-tailed Student’s t-test (A,B) or by one-way ANOVA with the Tukey post hoc test (C,D) (n = 3). ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus WT, or AdshLacZ; # p < 0.05 versus AdshScx. NS, not significant.

Figure 4

Loss of scleraxis attenuates TGFβ-induced glutaminolysis. (A–C) Cardiac fibroblasts were isolated from wild-type (WT) or scleraxis knockout (KO) mice, treated with vehicle or TGFβ₁ (10 ng/mL) for 24 h, and assayed for intracellular [glutamate + glutamine] (A), [glutamate] (B), and [glutamine] (C). Statistical analysis was by two-way ANOVA with the Tukey post hoc test (n = 3–4). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus WT + Vehicle.

Figure 5

Scleraxis transactivates the human GLS1 gene promoter. (A) Schematic depicting the proximal ~1.1 kilobase human GLS1 gene promoter containing three putative E-box sequences (E1–E3); numbers depict distance in nucleotides relative to the transcription start site. (B) NIH3T3 fibroblasts were transfected with an hGLS1 promoter luciferase reporter vector plus empty vector (pcDNA) or plus vector encoding scleraxis (pcDNA-Scx) and assayed for luciferase expression. (C) NIH3T3 fibroblasts were transfected as in (B), with the hGLS1 promoter with either intact (white fill) or sequentially mutated (black fill) E-boxes, and assayed for luciferase activity. (D) Chromatin immunoprecipitation was performed in human adult cardiac myofibroblasts using antibodies to scleraxis (Scx) or IgG control and primers spanning the region encompassing E1 to E3, with amplification carried out by qPCR. (E) Human cardiac myofibroblasts were treated with 10 ng/mL TGFβ and/or 0.3 μΜ CB-839 for 24 h, then assayed for α-SMA expression by qPCR. Statistical analysis was by one-way ANOVA with the Tukey post hoc test (B) (n = 3–4), or by the Kruskal–Wallis test followed by Dunn’s multiple comparisons test (C) (n = 3–4), or by the two-sided Student’s t-test (D) (n = 3), or by two-way ANOVA with Tukey (E) (n = 3). * p < 0.05, ** p < 0.01, **** p < 0.0001 versus pcDNA or versus IgG or versus vehicle; # p < 0.05, ## p < 0.01, #### p < 0.0001 versus samples as indicated (B) or versus TGFβ alone (E).

Figure 6

Scleraxis (red text) regulation of gene expression in fibrosis. Extracellular matrix (ECM) synthesis and processing require large quantities of ATP, which may be derived from various intracellular metabolic pathways, including fatty acid b-oxidation, glycolysis, glucose oxidation, and glutaminolysis. Key enzymes of each pathway are depicted, including those of glutaminolysis (green text). Red arrows highlight mechanisms that may contribute to cardiac fibrosis: scleraxis regulates GLS1 expression, per the present results, and shows evidence of regulation of other glutaminolysis enzyme genes. Scleraxis also directly transactivates a variety of pro-fibrotic genes, including ECM components Col1α2 and EDA-Fn; thus, scleraxis may regulate fibrosis by controlling both ECM gene expression and glutaminolysis genes to provide the energy necessary to support fibrosis.