Metabolic and Proliferative State of Vascular Adventitial Fibroblasts in Pulmonary Hypertension Is Regulated Through a MicroRNA-124/PTBP1 (Polypyrimidine Tract Binding Protein 1)/Pyruvate Kinase Muscle Axis

Abstract

Background: An emerging metabolic theory of pulmonary hypertension (PH) suggests that cellular and mitochondrial metabolic dysfunction underlies the pathology of this disease. We and others have previously demonstrated the existence of hyperproliferative, apoptosis-resistant, proinflammatory adventitial fibroblasts from human and bovine hypertensive pulmonary arterial walls (PH-Fibs) that exhibit constitutive reprogramming of glycolytic and mitochondrial metabolism, accompanied by an increased ratio of glucose catabolism through glycolysis versus the tricarboxylic acid cycle. However, the mechanisms responsible for these metabolic alterations in PH-Fibs remain unknown. We hypothesized that in PH-Fibs microRNA-124 (miR-124) regulates PTBP1 (polypyrimidine tract binding protein 1) expression to control alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2, resulting in an increased PKM2/PKM1 ratio, which promotes glycolysis and proliferation even in aerobic environments.

Methods: Pulmonary adventitial fibroblasts were isolated from calves and humans with severe PH (PH-Fibs) and from normal subjects. PTBP1 gene knockdown was achieved via PTBP1-siRNA; restoration of miR-124 was performed with miR-124 mimic. TEPP-46 and shikonin were used to manipulate PKM2 glycolytic function. Histone deacetylase inhibitors were used to treat cells. Metabolic products were determined by mass spectrometry-based metabolomics analyses, and mitochondrial function was analyzed by confocal microscopy and spectrofluorometry.

Results: We detected an increased PKM2/PKM1 ratio in PH-Fibs compared with normal subjects. PKM2 inhibition reversed the glycolytic status of PH-Fibs, decreased their cell proliferation, and attenuated macrophage interleukin-1β expression. Furthermore, normalizing the PKM2/PKM1 ratio in PH-Fibs by miR-124 overexpression or PTBP1 knockdown reversed the glycolytic phenotype (decreased the production of glycolytic intermediates and byproducts, ie, lactate), rescued mitochondrial reprogramming, and decreased cell proliferation. Pharmacological manipulation of PKM2 activity with TEPP-46 and shikonin or treatment with histone deacetylase inhibitors produced similar results.

Conclusions: In PH, miR-124, through the alternative splicing factor PTBP1, regulates the PKM2/PKM1 ratio, the overall metabolic, proliferative, and inflammatory state of cells. This PH phenotype can be rescued with interventions at various levels of the metabolic cascade. These findings suggest a more integrated view of vascular cell metabolism, which may open unique therapeutic prospects in targeting the dynamic glycolytic and mitochondrial interactions and between mesenchymal inflammatory cells in PH.

Keywords: TEEP-46; hypoxia; metabolism; mitochondria; pyruvate kinase; shikonin; splicing factors.

Fig. 1. The levels of PKM2 relative to PKM1 is increased in PH-Fibs compare to CO-Fibs, accompanied by decreased MPC and SIRT3 expression

Real-time RT-PCR analysis of PKM1 mRNA levels in CO- and PH-Fibs (A, bovine; I, human). PKM2/PKM1 mRNA ratio in CO- and PH-Fibs (B, bovine; J, human). Quantification of PKM1 protein levels (C, bovine; K, human) and PKM2/PKM1 protein ratio in Fibs (D, bovine; L, human) are presented. (Based on multiple WBs from several Fibs). Representative Western-blot of PKM1 and PKM2 protein expression in CO- and PH-Fibs (E, bovine; M, human). Real-time RT-PCR showed mRNA levels of MPC1 (F, bovine; N, human), MPC2 (G, bovine; O, human) and SIRT3 (H, bovine; P, human) in CO- and PH-Fibs. (Data are presented as mean ±S.E.M, n=4–8, *P<0.05 vs. CO-Fibs).

Fig. 2. PKM2 siRNA reduces PKM2 levels, PKM2/PKM1 ratio, lactate production and cell proliferation in human PH-Fibs

(A). Real-time RT-PCR analysis of PKM2 mRNA in human PH-Fibs targeted with PKM2 specific siRNA (siPKM2) or scrambled siRNA (SCR) (n=3, *P<0.05, compared to scrambled siRNA treated PH-Fibs) (B). PKM2/PKM1 mRNA ratio in human PH-Fibs targeted with SCR or siPKM2 (n=3, *P<0.05, compared to scrambled siRNA treated PH-Fibs). (C). Representative of Western-blot analysis of PKM2 and PKM1 protein expression in PH-Fibs targeted with SCR or PKM2 siRNAs (n=3). (D). UHPLC-MS analysis of lactate production in PH-Fibs targeted with SCR or siPKM2 after transfection (n=3, *P<0.05, compared to scrambled siRNA treated PH-Fibs). (E). Heat-map generated from MS (a detail of hierarchical clustering analysis) showed the levels of metabolites involved in glycolysis and mitochondrial oxidative phosphorylation post-siRNA transfection. (F) Data generated from MS (partial least-square discriminant analysis) showed siPKM2 altered the overall metabolic status of human PH-Fibs. (G) CyQUANT assay showed cell proliferation decreased by siPKM2. (n=3, *P<0.05, compared to scrambled siRNA treated PH-Fibs-repeated measures ANOVA).

Fig. 3. Treatment with TTEP-46 and Shikonin decreases lactate production and cell proliferation, and rescues mitochondrial bioenergetics in PH-Fibs

(A) Lactate production in human PH-Fibs after treatment by TEPP-46 (100 µM). (F) Glucose and lactate in human CO and PH-Fibs after treatment by Shikonin (0.5 µM and 1.0 µM) (n=3, *P<0.05, compared to DMSO treated PH-Fibs). (B, G) Heat-map generated from MS showed the levels of metabolites involved in glycolysis, serine biosynthesis and mitochondrial oxidative phosphorylation in human Fibs with treatment of TEPP-46 and Shikonin. (H) Overall metabolic phenotype of CO and PH-Fibs after DMSO or Shikonin treatment as gleaned by Partial Least Square-Discriminant analysis. (C, I) Proliferation assay showed human PH-Fibs proliferation after treatment by TEPP-46 and Shikonin. (n=3, *P<0.05, compared to DMSO treated PH-Fibs-repeated measures ANOVA). Mitochondrial respiratory control ratio (D) and maximal respiratory capacity (E) are presented in bovine PH-Fibs after TEPP-46 (100µM) treatment. (n=3, *P<0.05, compared to DMSO treated PH-Fibs).

Fig. 4. MiR-124 regulates PKM2/PKM1 expression through PTBP1 by alternative splicing

(A) Real-time RT-PCR showed overexpression of PTBP1 overrode the decreased PKM2/PKM1 ratio induced by miR-124 mimic in bovine PH-Fibs. (B) Representative western-blot of PTBP1, PKM1, and PKM2 in bovine PH-Fibs targeted with indicated treatments. (C) Schematic representation of a PKM splicing reporter driven by CMV promoter. Dashed lines indicate that primers are designed at the junctions of the exons. Arrows represent primers used for real-time RT-PCR. E9/E11 or E10/11, with Vector-R (located in vector sequence) for PKM1 or PKM2 transcripts expressed from the splicing reporter while Vector F with PKM total was used to quantify the total transcripts produced from the splicing reporter. The total transcript results were used to control transfection/expression efficiency of the splicing reporter in CO and PH-Fibs. (D) Relative PKM2 transcripts expressed from the splicing reporter in human CO-Fibs and PH-Fibs (with or without transfection of miR-124 mimic and siPTBP1) were presented. PKM2 levels were presented after calibrated with total PKM transcripts expressed from the splicing reporter to control for transfection and expression variation between CO and PH-Fibs. (n=3; *P<0.05).

Fig. 5. MiR-124 overexpression or PTBP1 silencing decreases the PKM2/PKM1 ratio and regulates the metabolic phenotype in PH-Fibs

Real-time RT-PCR showed PKM2/PKM1 mRNA ratio in CO-Fibs treated with scrambled siRNA (SCR) and PH-Fibs targeted with scrambled siRNA, miR-124 mimics or siPTBP1. (A, bovine; E, human, up panel). Representative Western blots of PKM1 and PKM2 are shown (A, bovine; E, human, low panel). Steady state UHPLC-MS demonstrated decreased lactate production in PH-Fibs post-transfection of miR-124 mimic or siPTBP1 (B, bovine; F, human). MS metabolomic analysis revealed that miR-124 mimic or siPTBP1 altered the overall metabolic status of both bovine (C) and human (G) PH-Fibs toward CO-Fib-like status. Heat-map generated from MS showed the levels of metabolites involved in glycolysis and mitochondrial oxidative phosphorylation post-transfection with miR-124 mimic or siPTBP1. Each column represents an individual sample from each group (D, bovine; H, human). (n=3, *P<0.05, compared to scrambled siRNA treated PH-Fibs).

Fig. 6. MiR-124 overexpression or PTBP1 silencing rescues mitochondrial alterations in bovine PH-Fibs

(A) Quantification of phosphorylation of PDH expressed as phosphorylated (P-PDH)/total PDH protein ratio in PH-Fibs transfected with miR-124 mimic or siPTBP1 (up panel). Representative western blots for phosphorylated PDH and total PDH in PH-Fibs are shown (low panel). Respiratory control ratio expressed as endogenous (state3)/ATP synthase inhibited (oligomycin, state4) respiratory ratio (B), maximal respiratory capacity expressed as uncoupled (FCCP)/ ATP synthase inhibited (oligomycin, state4) respiratory ratio (C), and in situ quantification of Complex I activity determined by rotenone-inhibited decrease of respiration (100% represents non-treated samples) (D) in CO-Fibs (CO-SCR) and PH-Fibs targeted with miR-124 mimics, or siPTBP1 are shown. (E) Quantification of NDUFS4 subunit of Complex I in PH-Fibs post-transfection by western blot analysis using specific NDUFS4 antibodies (up panel). Representative western blot is shown (low panel), including control of protein loading (Tim23 quantification). Mitochondrial membrane potential expressed by JC1 ratio (F), mitochondrial superoxide expressed by MitoSOX fluorescence increase rate Jm (G), and cytosolic ROS production detected using DCF probe (H) were revealed in CO-Fibs and PH-Fibs after transfection with miR-124 mimic or siPTBP1. (n=3–4, *P<0.05, compared to scrambled siRNA treated PH-Fibs).

Fig. 7. HDAC Inhibitors normalize the PKM2/PKM1 ratio and reverse the glycolytic metabolic phenotype of PH-Fibs

Real-time RT-PCR showed HDAC inhibitors decreased PKM2/PKM1 mRNA ratio (up panel; A, bovine; D, human). Representative western blot of PKM1 and PKM2 protein levels in these PH-Fibs are shown (low panel; A, bovine; D, human). Steady state UHPLC-MS demonstrated decreased glucose uptake and lactate production in PH-Fibs post-treatment of HDACi (B, bovine; E, human). Heat-map generated from MS showed the levels of metabolites involved in glycolysis, serine biosynthesis and mitochondrial oxidative phosphorylation in PH-Fibs post-treatment of HDACi. Each column represents an individual sample from each group (C, bovine; F, human). (n=3–4, *P<0.05, compared to DMSO treated PH-Fibs).

Fig. 8. HDAC Inhibitors rescue mitochondrial alterations in bovine PH-Fibs

HDACi treatment significantly increased respiratory control ratio (A), maximal respiratory capacity (B), Complex I activity (C) and NDUFS4 subunit of Complex I (D) in bovine PH-Fibs. Mitochondrial superoxide production (E) and cytosolic ROS production (F) decreased after HDACi treatment. (n=3–4, *P<0.05, compared to DMSO treated PH-Fibs). (G) Proposed mechanism contributing to metabolic reprogramming in pulmonary hypertensive fibroblasts. In PH-Fibs, the state of PKM isoform expression is controlled by an alternative splicing complex, composed of PTBP1, hnRNPA1 and hnRNPA2. In the presence of these PKM alternative splicing proteins, especially PTBP1 (a direct target of miR-124), exon 10 is included in the mature PKM transcript while exon 9 is excluded, resulting in a high PKM2/PKM1 ratio. The elevated PKM2/PKM1 ratio is critical for the constitutive reprogramming of glycolytic and mitochondrial metabolism, accompanied by an increased ratio of glucose catabolism through glycolysis versus the TCA cycle and increased ROS levels. PH-Fibs with an elevated PKM2/PKM1 ratio slow the production of pyruvate in response to pro-proliferative signaling enabling utilization of glycolytic intermediates for biosynthesis of cellular building blocks, which promote cell proliferation. Restoration of the PKM2/PKM1 ratio toward normal with miR-124 mimic and siPTBP1, or pharmacologic inhibition of PKM2 glycolytic function with TEPP-46 and Shikonin, or treatment with HDACi reverse glycolytic and mitochondrial reprograming toward normal. These findings provide additional mechanistic underpinnings for potentially targeting the miR-124-PTBP1-PKM axis as a novel therapeutic strategy in PH.